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[ "Japanese encephalitis virus (JEV), an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed.", "In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile.", "Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available.", "IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae.", "Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV). Most flaviviruses are arthropod borne and cause public health problems worldwide (1) .", "Most flaviviruses are arthropod borne and cause public health problems worldwide (1) . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus (TBEV), have decreased the rates of morbidity and mortality from infections caused by these viruses (2) ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini.", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini. The ORF encodes three structural proteins, including the capsid (C), membrane (premembrane [prM] and membrane [M] ), and envelope (E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (3) . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance.", "These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 (DENV-2). The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease.", "The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) .", "Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal (S/B) ratio, coefficient of variation (CV), and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig.", "A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected.", "After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) .", "1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine (DHP) voltage-gated Ca 2ϩ channel (VGCC) antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig.", "All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig. 1C) . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig.", "As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig. 2B) . A sharp decrease in JEV RNA levels was also detected (Fig. 2C) .", "A sharp decrease in JEV RNA levels was also detected (Fig. 2C) . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%.", "The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig.", "Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig. 2D) . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry.", "3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney (BHK-21) cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig.", "It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig. 3C) , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig.", "Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig. 4A) . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig.", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig. 4A) . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields.", "Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig.", "The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig. 4B and C). Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors.", "To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine (PAA) VGCC inhibitor (15) , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney (Vero) and human hepatocellular carcinoma (Huh-7) cells (Fig. 5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", "5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate (2-APB), which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum (ER) pool, respectively (16) (17) (18) (19) , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection.", "Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV.", "Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 (P20) showed robust resistance compared with the wild type (WT) (Fig. 6A ).", "6A ). When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified.", "Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine (Q)-to-arginine (R) switch at amino acid position 130 in transmembrane domain 3 (TMD3) of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone (Fig. 6B ).", "6B ). Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position (Fig. 6B) . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig.", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig. 4) , while YFV showed resistance to the drug (data not shown). To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses.", "To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig.", "Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig. 6C) , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine.", "6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine.", "In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs (Fig. 2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model.", "2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived).", "Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived). Manidipine treatment following JEV infection reduced the mortality rate to 20% (12 out of 15 animals survived) (Fig. 7A ).", "7A ). Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality.", "These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig.", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig. 7B) . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) .", "As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena (Fig. 7D) .", "7D) . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig.", "To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated.", "To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication.", "As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) .", "NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) .", "Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) .", "However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) .", "Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32.", "Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration (C max ) of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml (40) , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) .", "Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses.", "Cells and viruses. BHK-21, SH-SY5Y (human neuroblastoma), Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China.", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China. JEV replicon recombinant viral particles (RVPs) were generated as previously described (4, 5) . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells.", "ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay.", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities (2,500 to 12,500 cells per well) were infected with from 1.25 to 20 l RVPs (1 to 16 copies per well). The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) .", "The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) . Methyl-␤-cyclodextrin and dimethyl sulfoxide (DMSO) were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library.", "HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals (Houston, TX, USA). The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A .", "1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen.", "The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . The experimental timeline is depicted in Fig. 2A .", "The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) .", "To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) . Vero cells were infected with 20 l RVPs for 1 h (0 to 1 h). The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig.", "The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig. 3A) . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells.", "To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above (Fig. 3A) . Manidipine-resistant virus.", "3A) . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel.", "As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 (pMWJEAT), kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously (4) . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells.", "Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China).", "Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China). The mice were randomly divided into four groups (30 mice per group): a JEV-infected and vehicle (2% Tween 80 plus 5% DMSO in phosphate-buffered saline [PBS])-treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31.", "For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation.", "Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China)." ]

[ "Japanese encephalitis virus (JEV), an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed.", "In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile.", "Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available.", "IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae.", "Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV). Most flaviviruses are arthropod borne and cause public health problems worldwide (1) .", "Most flaviviruses are arthropod borne and cause public health problems worldwide (1) . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus (TBEV), have decreased the rates of morbidity and mortality from infections caused by these viruses (2) ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini.", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini. The ORF encodes three structural proteins, including the capsid (C), membrane (premembrane [prM] and membrane [M] ), and envelope (E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (3) . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance.", "These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 (DENV-2). The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease.", "The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) .", "Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal (S/B) ratio, coefficient of variation (CV), and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig.", "A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected.", "After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) .", "1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine (DHP) voltage-gated Ca 2ϩ channel (VGCC) antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig.", "All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig. 1C) . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig.", "As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig. 2B) . A sharp decrease in JEV RNA levels was also detected (Fig. 2C) .", "A sharp decrease in JEV RNA levels was also detected (Fig. 2C) . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%.", "The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig.", "Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig. 2D) . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry.", "3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney (BHK-21) cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig.", "It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig. 3C) , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig.", "Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig. 4A) . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig.", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig. 4A) . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields.", "Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig.", "The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig. 4B and C). Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors.", "To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine (PAA) VGCC inhibitor (15) , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney (Vero) and human hepatocellular carcinoma (Huh-7) cells (Fig. 5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", "5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate (2-APB), which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum (ER) pool, respectively (16) (17) (18) (19) , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection.", "Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV.", "Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 (P20) showed robust resistance compared with the wild type (WT) (Fig. 6A ).", "6A ). When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified.", "Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine (Q)-to-arginine (R) switch at amino acid position 130 in transmembrane domain 3 (TMD3) of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone (Fig. 6B ).", "6B ). Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position (Fig. 6B) . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig.", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig. 4) , while YFV showed resistance to the drug (data not shown). To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses.", "To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig.", "Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig. 6C) , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine.", "6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine.", "In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs (Fig. 2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model.", "2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived).", "Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived). Manidipine treatment following JEV infection reduced the mortality rate to 20% (12 out of 15 animals survived) (Fig. 7A ).", "7A ). Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality.", "These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig.", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig. 7B) . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) .", "As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena (Fig. 7D) .", "7D) . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig.", "To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated.", "To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication.", "As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) .", "NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) .", "Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) .", "However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) .", "Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32.", "Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration (C max ) of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml (40) , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) .", "Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses.", "Cells and viruses. BHK-21, SH-SY5Y (human neuroblastoma), Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China.", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China. JEV replicon recombinant viral particles (RVPs) were generated as previously described (4, 5) . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells.", "ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay.", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities (2,500 to 12,500 cells per well) were infected with from 1.25 to 20 l RVPs (1 to 16 copies per well). The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) .", "The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) . Methyl-␤-cyclodextrin and dimethyl sulfoxide (DMSO) were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library.", "HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals (Houston, TX, USA). The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A .", "1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen.", "The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . The experimental timeline is depicted in Fig. 2A .", "The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) .", "To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) . Vero cells were infected with 20 l RVPs for 1 h (0 to 1 h). The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig.", "The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig. 3A) . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells.", "To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above (Fig. 3A) . Manidipine-resistant virus.", "3A) . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel.", "As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 (pMWJEAT), kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously (4) . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells.", "Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China).", "Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China). The mice were randomly divided into four groups (30 mice per group): a JEV-infected and vehicle (2% Tween 80 plus 5% DMSO in phosphate-buffered saline [PBS])-treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31.", "For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation.", "Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China)." ]

[ "Japanese encephalitis virus (JEV), an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed.", "In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile.", "Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available.", "IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae.", "Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV). Most flaviviruses are arthropod borne and cause public health problems worldwide (1) .", "Most flaviviruses are arthropod borne and cause public health problems worldwide (1) . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus (TBEV), have decreased the rates of morbidity and mortality from infections caused by these viruses (2) ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini.", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini. The ORF encodes three structural proteins, including the capsid (C), membrane (premembrane [prM] and membrane [M] ), and envelope (E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (3) . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance.", "These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 (DENV-2). The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease.", "The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) .", "Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal (S/B) ratio, coefficient of variation (CV), and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig.", "A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected.", "After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) .", "1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine (DHP) voltage-gated Ca 2ϩ channel (VGCC) antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig.", "All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig. 1C) . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig.", "As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig. 2B) . A sharp decrease in JEV RNA levels was also detected (Fig. 2C) .", "A sharp decrease in JEV RNA levels was also detected (Fig. 2C) . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%.", "The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig.", "Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig. 2D) . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry.", "3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney (BHK-21) cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig.", "It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig. 3C) , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig.", "Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig. 4A) . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig.", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig. 4A) . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields.", "Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig.", "The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig. 4B and C). Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors.", "To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine (PAA) VGCC inhibitor (15) , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney (Vero) and human hepatocellular carcinoma (Huh-7) cells (Fig. 5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", "5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate (2-APB), which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum (ER) pool, respectively (16) (17) (18) (19) , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection.", "Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV.", "Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 (P20) showed robust resistance compared with the wild type (WT) (Fig. 6A ).", "6A ). When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified.", "Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine (Q)-to-arginine (R) switch at amino acid position 130 in transmembrane domain 3 (TMD3) of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone (Fig. 6B ).", "6B ). Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position (Fig. 6B) . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig.", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig. 4) , while YFV showed resistance to the drug (data not shown). To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses.", "To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig.", "Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig. 6C) , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine.", "6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine.", "In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs (Fig. 2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model.", "2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived).", "Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived). Manidipine treatment following JEV infection reduced the mortality rate to 20% (12 out of 15 animals survived) (Fig. 7A ).", "7A ). Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality.", "These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig.", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig. 7B) . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) .", "As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena (Fig. 7D) .", "7D) . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig.", "To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated.", "To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication.", "As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) .", "NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) .", "Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) .", "However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) .", "Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32.", "Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration (C max ) of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml (40) , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) .", "Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses.", "Cells and viruses. BHK-21, SH-SY5Y (human neuroblastoma), Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China.", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China. JEV replicon recombinant viral particles (RVPs) were generated as previously described (4, 5) . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells.", "ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay.", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities (2,500 to 12,500 cells per well) were infected with from 1.25 to 20 l RVPs (1 to 16 copies per well). The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) .", "The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) . Methyl-␤-cyclodextrin and dimethyl sulfoxide (DMSO) were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library.", "HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals (Houston, TX, USA). The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A .", "1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen.", "The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . The experimental timeline is depicted in Fig. 2A .", "The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) .", "To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) . Vero cells were infected with 20 l RVPs for 1 h (0 to 1 h). The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig.", "The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig. 3A) . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells.", "To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above (Fig. 3A) . Manidipine-resistant virus.", "3A) . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel.", "As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 (pMWJEAT), kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously (4) . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells.", "Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China).", "Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China). The mice were randomly divided into four groups (30 mice per group): a JEV-infected and vehicle (2% Tween 80 plus 5% DMSO in phosphate-buffered saline [PBS])-treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31.", "For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation.", "Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China)." ]

[ "Japanese encephalitis virus (JEV), an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed.", "In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile.", "Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available.", "IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae.", "Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV). Most flaviviruses are arthropod borne and cause public health problems worldwide (1) .", "Most flaviviruses are arthropod borne and cause public health problems worldwide (1) . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus (TBEV), have decreased the rates of morbidity and mortality from infections caused by these viruses (2) ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini.", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini. The ORF encodes three structural proteins, including the capsid (C), membrane (premembrane [prM] and membrane [M] ), and envelope (E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (3) . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance.", "These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 (DENV-2). The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease.", "The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) .", "Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal (S/B) ratio, coefficient of variation (CV), and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig.", "A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected.", "After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) .", "1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine (DHP) voltage-gated Ca 2ϩ channel (VGCC) antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig.", "All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig. 1C) . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig.", "As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig. 2B) . A sharp decrease in JEV RNA levels was also detected (Fig. 2C) .", "A sharp decrease in JEV RNA levels was also detected (Fig. 2C) . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%.", "The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig.", "Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig. 2D) . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry.", "3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney (BHK-21) cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig.", "It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig. 3C) , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig.", "Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig. 4A) . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig.", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig. 4A) . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields.", "Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig.", "The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig. 4B and C). Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors.", "To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine (PAA) VGCC inhibitor (15) , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney (Vero) and human hepatocellular carcinoma (Huh-7) cells (Fig. 5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", "5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate (2-APB), which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum (ER) pool, respectively (16) (17) (18) (19) , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection.", "Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV.", "Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 (P20) showed robust resistance compared with the wild type (WT) (Fig. 6A ).", "6A ). When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified.", "Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine (Q)-to-arginine (R) switch at amino acid position 130 in transmembrane domain 3 (TMD3) of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone (Fig. 6B ).", "6B ). Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position (Fig. 6B) . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig.", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig. 4) , while YFV showed resistance to the drug (data not shown). To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses.", "To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig.", "Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig. 6C) , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine.", "6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine.", "In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs (Fig. 2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model.", "2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived).", "Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived). Manidipine treatment following JEV infection reduced the mortality rate to 20% (12 out of 15 animals survived) (Fig. 7A ).", "7A ). Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality.", "These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig.", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig. 7B) . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) .", "As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena (Fig. 7D) .", "7D) . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig.", "To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated.", "To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication.", "As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) .", "NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) .", "Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) .", "However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) .", "Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32.", "Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration (C max ) of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml (40) , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) .", "Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses.", "Cells and viruses. BHK-21, SH-SY5Y (human neuroblastoma), Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China.", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China. JEV replicon recombinant viral particles (RVPs) were generated as previously described (4, 5) . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells.", "ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay.", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities (2,500 to 12,500 cells per well) were infected with from 1.25 to 20 l RVPs (1 to 16 copies per well). The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) .", "The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) . Methyl-␤-cyclodextrin and dimethyl sulfoxide (DMSO) were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library.", "HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals (Houston, TX, USA). The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A .", "1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen.", "The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . The experimental timeline is depicted in Fig. 2A .", "The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) .", "To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) . Vero cells were infected with 20 l RVPs for 1 h (0 to 1 h). The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig.", "The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig. 3A) . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells.", "To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above (Fig. 3A) . Manidipine-resistant virus.", "3A) . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel.", "As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 (pMWJEAT), kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously (4) . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells.", "Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China).", "Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China). The mice were randomly divided into four groups (30 mice per group): a JEV-infected and vehicle (2% Tween 80 plus 5% DMSO in phosphate-buffered saline [PBS])-treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31.", "For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation.", "Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China)." ]

[ "Japanese encephalitis virus (JEV), an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed.", "In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile.", "Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available.", "IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae.", "Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV). Most flaviviruses are arthropod borne and cause public health problems worldwide (1) .", "Most flaviviruses are arthropod borne and cause public health problems worldwide (1) . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus (TBEV), have decreased the rates of morbidity and mortality from infections caused by these viruses (2) ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini.", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini. The ORF encodes three structural proteins, including the capsid (C), membrane (premembrane [prM] and membrane [M] ), and envelope (E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (3) . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance.", "These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 (DENV-2). The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease.", "The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) .", "Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal (S/B) ratio, coefficient of variation (CV), and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig.", "A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected.", "After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) .", "1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine (DHP) voltage-gated Ca 2ϩ channel (VGCC) antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig.", "All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig. 1C) . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig.", "As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig. 2B) . A sharp decrease in JEV RNA levels was also detected (Fig. 2C) .", "A sharp decrease in JEV RNA levels was also detected (Fig. 2C) . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%.", "The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig.", "Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig. 2D) . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry.", "3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney (BHK-21) cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig.", "It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig. 3C) , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig.", "Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig. 4A) . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig.", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig. 4A) . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields.", "Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig.", "The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig. 4B and C). Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors.", "To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine (PAA) VGCC inhibitor (15) , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney (Vero) and human hepatocellular carcinoma (Huh-7) cells (Fig. 5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", "5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate (2-APB), which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum (ER) pool, respectively (16) (17) (18) (19) , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection.", "Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV.", "Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 (P20) showed robust resistance compared with the wild type (WT) (Fig. 6A ).", "6A ). When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified.", "Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine (Q)-to-arginine (R) switch at amino acid position 130 in transmembrane domain 3 (TMD3) of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone (Fig. 6B ).", "6B ). Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position (Fig. 6B) . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig.", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig. 4) , while YFV showed resistance to the drug (data not shown). To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses.", "To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig.", "Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig. 6C) , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine.", "6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine.", "In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs (Fig. 2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model.", "2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived).", "Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived). Manidipine treatment following JEV infection reduced the mortality rate to 20% (12 out of 15 animals survived) (Fig. 7A ).", "7A ). Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality.", "These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig.", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig. 7B) . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) .", "As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena (Fig. 7D) .", "7D) . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig.", "To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated.", "To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication.", "As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) .", "NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) .", "Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) .", "However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) .", "Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32.", "Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration (C max ) of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml (40) , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) .", "Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses.", "Cells and viruses. BHK-21, SH-SY5Y (human neuroblastoma), Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China.", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China. JEV replicon recombinant viral particles (RVPs) were generated as previously described (4, 5) . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells.", "ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay.", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities (2,500 to 12,500 cells per well) were infected with from 1.25 to 20 l RVPs (1 to 16 copies per well). The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) .", "The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) . Methyl-␤-cyclodextrin and dimethyl sulfoxide (DMSO) were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library.", "HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals (Houston, TX, USA). The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A .", "1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen.", "The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . The experimental timeline is depicted in Fig. 2A .", "The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) .", "To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) . Vero cells were infected with 20 l RVPs for 1 h (0 to 1 h). The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig.", "The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig. 3A) . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells.", "To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above (Fig. 3A) . Manidipine-resistant virus.", "3A) . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel.", "As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 (pMWJEAT), kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously (4) . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells.", "Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China).", "Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China). The mice were randomly divided into four groups (30 mice per group): a JEV-infected and vehicle (2% Tween 80 plus 5% DMSO in phosphate-buffered saline [PBS])-treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31.", "For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation.", "Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China)." ]

[ "Japanese encephalitis virus (JEV), an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed.", "In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile.", "Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available.", "IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae.", "Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV). Most flaviviruses are arthropod borne and cause public health problems worldwide (1) .", "Most flaviviruses are arthropod borne and cause public health problems worldwide (1) . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus (TBEV), have decreased the rates of morbidity and mortality from infections caused by these viruses (2) ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini.", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini. The ORF encodes three structural proteins, including the capsid (C), membrane (premembrane [prM] and membrane [M] ), and envelope (E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (3) . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance.", "These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 (DENV-2). The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease.", "The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) .", "Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal (S/B) ratio, coefficient of variation (CV), and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig.", "A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected.", "After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) .", "1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine (DHP) voltage-gated Ca 2ϩ channel (VGCC) antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig.", "All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig. 1C) . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig.", "As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig. 2B) . A sharp decrease in JEV RNA levels was also detected (Fig. 2C) .", "A sharp decrease in JEV RNA levels was also detected (Fig. 2C) . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%.", "The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig.", "Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig. 2D) . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry.", "3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney (BHK-21) cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig.", "It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig. 3C) , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig.", "Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig. 4A) . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig.", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig. 4A) . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields.", "Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig.", "The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig. 4B and C). Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors.", "To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine (PAA) VGCC inhibitor (15) , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney (Vero) and human hepatocellular carcinoma (Huh-7) cells (Fig. 5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", "5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate (2-APB), which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum (ER) pool, respectively (16) (17) (18) (19) , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection.", "Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV.", "Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 (P20) showed robust resistance compared with the wild type (WT) (Fig. 6A ).", "6A ). When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified.", "Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine (Q)-to-arginine (R) switch at amino acid position 130 in transmembrane domain 3 (TMD3) of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone (Fig. 6B ).", "6B ). Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position (Fig. 6B) . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig.", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig. 4) , while YFV showed resistance to the drug (data not shown). To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses.", "To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig.", "Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig. 6C) , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine.", "6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine.", "In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs (Fig. 2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model.", "2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived).", "Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived). Manidipine treatment following JEV infection reduced the mortality rate to 20% (12 out of 15 animals survived) (Fig. 7A ).", "7A ). Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality.", "These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig.", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig. 7B) . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) .", "As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena (Fig. 7D) .", "7D) . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig.", "To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated.", "To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication.", "As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) .", "NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) .", "Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) .", "However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) .", "Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32.", "Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration (C max ) of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml (40) , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) .", "Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses.", "Cells and viruses. BHK-21, SH-SY5Y (human neuroblastoma), Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China.", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China. JEV replicon recombinant viral particles (RVPs) were generated as previously described (4, 5) . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells.", "ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay.", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities (2,500 to 12,500 cells per well) were infected with from 1.25 to 20 l RVPs (1 to 16 copies per well). The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) .", "The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) . Methyl-␤-cyclodextrin and dimethyl sulfoxide (DMSO) were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library.", "HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals (Houston, TX, USA). The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A .", "1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen.", "The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . The experimental timeline is depicted in Fig. 2A .", "The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) .", "To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) . Vero cells were infected with 20 l RVPs for 1 h (0 to 1 h). The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig.", "The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig. 3A) . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells.", "To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above (Fig. 3A) . Manidipine-resistant virus.", "3A) . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel.", "As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 (pMWJEAT), kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously (4) . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells.", "Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China).", "Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China). The mice were randomly divided into four groups (30 mice per group): a JEV-infected and vehicle (2% Tween 80 plus 5% DMSO in phosphate-buffered saline [PBS])-treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31.", "For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation.", "Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China)." ]

[ "Japanese encephalitis virus (JEV), an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed.", "In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile.", "Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available.", "IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae.", "Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV). Most flaviviruses are arthropod borne and cause public health problems worldwide (1) .", "Most flaviviruses are arthropod borne and cause public health problems worldwide (1) . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus (TBEV), have decreased the rates of morbidity and mortality from infections caused by these viruses (2) ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini.", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini. The ORF encodes three structural proteins, including the capsid (C), membrane (premembrane [prM] and membrane [M] ), and envelope (E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (3) . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance.", "These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 (DENV-2). The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease.", "The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) .", "Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal (S/B) ratio, coefficient of variation (CV), and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig.", "A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected.", "After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) .", "1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine (DHP) voltage-gated Ca 2ϩ channel (VGCC) antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig.", "All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig. 1C) . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig.", "As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig. 2B) . A sharp decrease in JEV RNA levels was also detected (Fig. 2C) .", "A sharp decrease in JEV RNA levels was also detected (Fig. 2C) . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%.", "The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig.", "Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig. 2D) . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry.", "3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney (BHK-21) cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig.", "It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig. 3C) , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig.", "Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig. 4A) . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig.", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig. 4A) . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields.", "Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig.", "The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig. 4B and C). Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors.", "To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine (PAA) VGCC inhibitor (15) , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney (Vero) and human hepatocellular carcinoma (Huh-7) cells (Fig. 5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", "5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate (2-APB), which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum (ER) pool, respectively (16) (17) (18) (19) , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection.", "Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV.", "Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 (P20) showed robust resistance compared with the wild type (WT) (Fig. 6A ).", "6A ). When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified.", "Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine (Q)-to-arginine (R) switch at amino acid position 130 in transmembrane domain 3 (TMD3) of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone (Fig. 6B ).", "6B ). Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position (Fig. 6B) . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig.", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig. 4) , while YFV showed resistance to the drug (data not shown). To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses.", "To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig.", "Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig. 6C) , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine.", "6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine.", "In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs (Fig. 2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model.", "2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived).", "Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived). Manidipine treatment following JEV infection reduced the mortality rate to 20% (12 out of 15 animals survived) (Fig. 7A ).", "7A ). Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality.", "These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig.", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig. 7B) . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) .", "As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena (Fig. 7D) .", "7D) . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig.", "To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated.", "To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication.", "As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) .", "NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) .", "Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) .", "However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) .", "Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32.", "Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration (C max ) of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml (40) , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) .", "Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses.", "Cells and viruses. BHK-21, SH-SY5Y (human neuroblastoma), Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China.", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China. JEV replicon recombinant viral particles (RVPs) were generated as previously described (4, 5) . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells.", "ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay.", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities (2,500 to 12,500 cells per well) were infected with from 1.25 to 20 l RVPs (1 to 16 copies per well). The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) .", "The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) . Methyl-␤-cyclodextrin and dimethyl sulfoxide (DMSO) were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library.", "HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals (Houston, TX, USA). The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A .", "1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen.", "The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . The experimental timeline is depicted in Fig. 2A .", "The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) .", "To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) . Vero cells were infected with 20 l RVPs for 1 h (0 to 1 h). The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig.", "The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig. 3A) . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells.", "To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above (Fig. 3A) . Manidipine-resistant virus.", "3A) . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel.", "As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 (pMWJEAT), kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously (4) . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells.", "Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China).", "Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China). The mice were randomly divided into four groups (30 mice per group): a JEV-infected and vehicle (2% Tween 80 plus 5% DMSO in phosphate-buffered saline [PBS])-treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31.", "For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation.", "Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China)." ]

[ "Japanese encephalitis virus (JEV), an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed.", "In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile.", "Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available.", "IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae.", "Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV). Most flaviviruses are arthropod borne and cause public health problems worldwide (1) .", "Most flaviviruses are arthropod borne and cause public health problems worldwide (1) . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus (TBEV), have decreased the rates of morbidity and mortality from infections caused by these viruses (2) ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini.", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini. The ORF encodes three structural proteins, including the capsid (C), membrane (premembrane [prM] and membrane [M] ), and envelope (E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (3) . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance.", "These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 (DENV-2). The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease.", "The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) .", "Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal (S/B) ratio, coefficient of variation (CV), and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig.", "A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected.", "After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) .", "1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine (DHP) voltage-gated Ca 2ϩ channel (VGCC) antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig.", "All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig. 1C) . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig.", "As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig. 2B) . A sharp decrease in JEV RNA levels was also detected (Fig. 2C) .", "A sharp decrease in JEV RNA levels was also detected (Fig. 2C) . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%.", "The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig.", "Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig. 2D) . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry.", "3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney (BHK-21) cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig.", "It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig. 3C) , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig.", "Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig. 4A) . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig.", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig. 4A) . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields.", "Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig.", "The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig. 4B and C). Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors.", "To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine (PAA) VGCC inhibitor (15) , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney (Vero) and human hepatocellular carcinoma (Huh-7) cells (Fig. 5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", "5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate (2-APB), which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum (ER) pool, respectively (16) (17) (18) (19) , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection.", "Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV.", "Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 (P20) showed robust resistance compared with the wild type (WT) (Fig. 6A ).", "6A ). When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified.", "Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine (Q)-to-arginine (R) switch at amino acid position 130 in transmembrane domain 3 (TMD3) of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone (Fig. 6B ).", "6B ). Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position (Fig. 6B) . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig.", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig. 4) , while YFV showed resistance to the drug (data not shown). To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses.", "To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig.", "Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig. 6C) , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine.", "6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine.", "In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs (Fig. 2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model.", "2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived).", "Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived). Manidipine treatment following JEV infection reduced the mortality rate to 20% (12 out of 15 animals survived) (Fig. 7A ).", "7A ). Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality.", "These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig.", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig. 7B) . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) .", "As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena (Fig. 7D) .", "7D) . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig.", "To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated.", "To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication.", "As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) .", "NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) .", "Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) .", "However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) .", "Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32.", "Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration (C max ) of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml (40) , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) .", "Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses.", "Cells and viruses. BHK-21, SH-SY5Y (human neuroblastoma), Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China.", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China. JEV replicon recombinant viral particles (RVPs) were generated as previously described (4, 5) . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells.", "ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay.", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities (2,500 to 12,500 cells per well) were infected with from 1.25 to 20 l RVPs (1 to 16 copies per well). The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) .", "The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) . Methyl-␤-cyclodextrin and dimethyl sulfoxide (DMSO) were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library.", "HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals (Houston, TX, USA). The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A .", "1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen.", "The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . The experimental timeline is depicted in Fig. 2A .", "The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) .", "To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) . Vero cells were infected with 20 l RVPs for 1 h (0 to 1 h). The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig.", "The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig. 3A) . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells.", "To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above (Fig. 3A) . Manidipine-resistant virus.", "3A) . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel.", "As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 (pMWJEAT), kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously (4) . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells.", "Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China).", "Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China). The mice were randomly divided into four groups (30 mice per group): a JEV-infected and vehicle (2% Tween 80 plus 5% DMSO in phosphate-buffered saline [PBS])-treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31.", "For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation.", "Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China)." ]

[ "Japanese encephalitis virus (JEV), an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed.", "In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile.", "Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available.", "IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae.", "Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV). Most flaviviruses are arthropod borne and cause public health problems worldwide (1) .", "Most flaviviruses are arthropod borne and cause public health problems worldwide (1) . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus (TBEV), have decreased the rates of morbidity and mortality from infections caused by these viruses (2) ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini.", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini. The ORF encodes three structural proteins, including the capsid (C), membrane (premembrane [prM] and membrane [M] ), and envelope (E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (3) . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance.", "These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 (DENV-2). The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease.", "The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) .", "Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal (S/B) ratio, coefficient of variation (CV), and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig.", "A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected.", "After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) .", "1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine (DHP) voltage-gated Ca 2ϩ channel (VGCC) antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig.", "All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig. 1C) . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig.", "As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig. 2B) . A sharp decrease in JEV RNA levels was also detected (Fig. 2C) .", "A sharp decrease in JEV RNA levels was also detected (Fig. 2C) . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%.", "The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig.", "Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig. 2D) . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry.", "3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney (BHK-21) cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig.", "It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig. 3C) , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig.", "Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig. 4A) . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig.", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig. 4A) . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields.", "Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig.", "The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig. 4B and C). Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors.", "To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine (PAA) VGCC inhibitor (15) , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney (Vero) and human hepatocellular carcinoma (Huh-7) cells (Fig. 5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", "5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate (2-APB), which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum (ER) pool, respectively (16) (17) (18) (19) , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection.", "Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV.", "Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 (P20) showed robust resistance compared with the wild type (WT) (Fig. 6A ).", "6A ). When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified.", "Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine (Q)-to-arginine (R) switch at amino acid position 130 in transmembrane domain 3 (TMD3) of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone (Fig. 6B ).", "6B ). Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position (Fig. 6B) . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig.", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig. 4) , while YFV showed resistance to the drug (data not shown). To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses.", "To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig.", "Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig. 6C) , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine.", "6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine.", "In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs (Fig. 2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model.", "2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived).", "Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived). Manidipine treatment following JEV infection reduced the mortality rate to 20% (12 out of 15 animals survived) (Fig. 7A ).", "7A ). Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality.", "These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig.", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig. 7B) . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) .", "As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena (Fig. 7D) .", "7D) . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig.", "To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated.", "To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication.", "As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) .", "NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) .", "Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) .", "However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) .", "Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32.", "Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration (C max ) of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml (40) , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) .", "Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses.", "Cells and viruses. BHK-21, SH-SY5Y (human neuroblastoma), Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China.", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China. JEV replicon recombinant viral particles (RVPs) were generated as previously described (4, 5) . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells.", "ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay.", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities (2,500 to 12,500 cells per well) were infected with from 1.25 to 20 l RVPs (1 to 16 copies per well). The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) .", "The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) . Methyl-␤-cyclodextrin and dimethyl sulfoxide (DMSO) were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library.", "HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals (Houston, TX, USA). The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A .", "1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen.", "The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . The experimental timeline is depicted in Fig. 2A .", "The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) .", "To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) . Vero cells were infected with 20 l RVPs for 1 h (0 to 1 h). The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig.", "The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig. 3A) . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells.", "To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above (Fig. 3A) . Manidipine-resistant virus.", "3A) . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel.", "As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 (pMWJEAT), kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously (4) . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells.", "Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China).", "Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China). The mice were randomly divided into four groups (30 mice per group): a JEV-infected and vehicle (2% Tween 80 plus 5% DMSO in phosphate-buffered saline [PBS])-treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31.", "For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation.", "Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China)." ]

[ "Japanese encephalitis virus (JEV), an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed.", "In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile.", "Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available.", "IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae.", "Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV). Most flaviviruses are arthropod borne and cause public health problems worldwide (1) .", "Most flaviviruses are arthropod borne and cause public health problems worldwide (1) . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus (TBEV), have decreased the rates of morbidity and mortality from infections caused by these viruses (2) ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini.", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini. The ORF encodes three structural proteins, including the capsid (C), membrane (premembrane [prM] and membrane [M] ), and envelope (E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (3) . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance.", "These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 (DENV-2). The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease.", "The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) .", "Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal (S/B) ratio, coefficient of variation (CV), and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig.", "A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected.", "After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) .", "1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine (DHP) voltage-gated Ca 2ϩ channel (VGCC) antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig.", "All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig. 1C) . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig.", "As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig. 2B) . A sharp decrease in JEV RNA levels was also detected (Fig. 2C) .", "A sharp decrease in JEV RNA levels was also detected (Fig. 2C) . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%.", "The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig.", "Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig. 2D) . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry.", "3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney (BHK-21) cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig.", "It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig. 3C) , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig.", "Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig. 4A) . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig.", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig. 4A) . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields.", "Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig.", "The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig. 4B and C). Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors.", "To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine (PAA) VGCC inhibitor (15) , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney (Vero) and human hepatocellular carcinoma (Huh-7) cells (Fig. 5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", "5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate (2-APB), which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum (ER) pool, respectively (16) (17) (18) (19) , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection.", "Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV.", "Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 (P20) showed robust resistance compared with the wild type (WT) (Fig. 6A ).", "6A ). When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified.", "Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine (Q)-to-arginine (R) switch at amino acid position 130 in transmembrane domain 3 (TMD3) of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone (Fig. 6B ).", "6B ). Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position (Fig. 6B) . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig.", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig. 4) , while YFV showed resistance to the drug (data not shown). To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses.", "To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig.", "Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig. 6C) , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine.", "6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine.", "In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs (Fig. 2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model.", "2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived).", "Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived). Manidipine treatment following JEV infection reduced the mortality rate to 20% (12 out of 15 animals survived) (Fig. 7A ).", "7A ). Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality.", "These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig.", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig. 7B) . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) .", "As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena (Fig. 7D) .", "7D) . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig.", "To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated.", "To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication.", "As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) .", "NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) .", "Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) .", "However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) .", "Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32.", "Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration (C max ) of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml (40) , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) .", "Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses.", "Cells and viruses. BHK-21, SH-SY5Y (human neuroblastoma), Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China.", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China. JEV replicon recombinant viral particles (RVPs) were generated as previously described (4, 5) . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells.", "ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay.", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities (2,500 to 12,500 cells per well) were infected with from 1.25 to 20 l RVPs (1 to 16 copies per well). The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) .", "The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) . Methyl-␤-cyclodextrin and dimethyl sulfoxide (DMSO) were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library.", "HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals (Houston, TX, USA). The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A .", "1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen.", "The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . The experimental timeline is depicted in Fig. 2A .", "The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) .", "To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) . Vero cells were infected with 20 l RVPs for 1 h (0 to 1 h). The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig.", "The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig. 3A) . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells.", "To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above (Fig. 3A) . Manidipine-resistant virus.", "3A) . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel.", "As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 (pMWJEAT), kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously (4) . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells.", "Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China).", "Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China). The mice were randomly divided into four groups (30 mice per group): a JEV-infected and vehicle (2% Tween 80 plus 5% DMSO in phosphate-buffered saline [PBS])-treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31.", "For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation.", "Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China)." ]

[ "Japanese encephalitis virus (JEV), an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed.", "In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile.", "Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available.", "IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae.", "Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV). Most flaviviruses are arthropod borne and cause public health problems worldwide (1) .", "Most flaviviruses are arthropod borne and cause public health problems worldwide (1) . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus (TBEV), have decreased the rates of morbidity and mortality from infections caused by these viruses (2) ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini.", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini. The ORF encodes three structural proteins, including the capsid (C), membrane (premembrane [prM] and membrane [M] ), and envelope (E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (3) . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance.", "These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 (DENV-2). The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease.", "The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) .", "Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal (S/B) ratio, coefficient of variation (CV), and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig.", "A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected.", "After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) .", "1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine (DHP) voltage-gated Ca 2ϩ channel (VGCC) antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig.", "All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig. 1C) . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig.", "As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig. 2B) . A sharp decrease in JEV RNA levels was also detected (Fig. 2C) .", "A sharp decrease in JEV RNA levels was also detected (Fig. 2C) . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%.", "The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig.", "Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig. 2D) . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry.", "3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney (BHK-21) cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig.", "It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig. 3C) , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig.", "Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig. 4A) . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig.", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig. 4A) . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields.", "Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig.", "The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig. 4B and C). Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors.", "To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine (PAA) VGCC inhibitor (15) , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney (Vero) and human hepatocellular carcinoma (Huh-7) cells (Fig. 5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", "5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate (2-APB), which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum (ER) pool, respectively (16) (17) (18) (19) , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection.", "Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV.", "Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 (P20) showed robust resistance compared with the wild type (WT) (Fig. 6A ).", "6A ). When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified.", "Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine (Q)-to-arginine (R) switch at amino acid position 130 in transmembrane domain 3 (TMD3) of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone (Fig. 6B ).", "6B ). Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position (Fig. 6B) . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig.", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig. 4) , while YFV showed resistance to the drug (data not shown). To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses.", "To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig.", "Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig. 6C) , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine.", "6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine.", "In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs (Fig. 2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model.", "2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived).", "Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived). Manidipine treatment following JEV infection reduced the mortality rate to 20% (12 out of 15 animals survived) (Fig. 7A ).", "7A ). Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality.", "These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig.", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig. 7B) . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) .", "As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena (Fig. 7D) .", "7D) . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig.", "To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated.", "To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication.", "As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) .", "NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) .", "Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) .", "However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) .", "Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32.", "Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration (C max ) of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml (40) , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) .", "Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses.", "Cells and viruses. BHK-21, SH-SY5Y (human neuroblastoma), Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China.", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China. JEV replicon recombinant viral particles (RVPs) were generated as previously described (4, 5) . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells.", "ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay.", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities (2,500 to 12,500 cells per well) were infected with from 1.25 to 20 l RVPs (1 to 16 copies per well). The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) .", "The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) . Methyl-␤-cyclodextrin and dimethyl sulfoxide (DMSO) were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library.", "HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals (Houston, TX, USA). The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A .", "1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen.", "The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . The experimental timeline is depicted in Fig. 2A .", "The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) .", "To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) . Vero cells were infected with 20 l RVPs for 1 h (0 to 1 h). The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig.", "The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig. 3A) . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells.", "To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above (Fig. 3A) . Manidipine-resistant virus.", "3A) . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel.", "As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 (pMWJEAT), kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously (4) . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells.", "Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China).", "Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China). The mice were randomly divided into four groups (30 mice per group): a JEV-infected and vehicle (2% Tween 80 plus 5% DMSO in phosphate-buffered saline [PBS])-treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31.", "For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation.", "Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China)." ]

[ "Japanese encephalitis virus (JEV), an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed.", "In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile.", "Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available.", "IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae.", "Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV). Most flaviviruses are arthropod borne and cause public health problems worldwide (1) .", "Most flaviviruses are arthropod borne and cause public health problems worldwide (1) . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus (TBEV), have decreased the rates of morbidity and mortality from infections caused by these viruses (2) ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini.", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini. The ORF encodes three structural proteins, including the capsid (C), membrane (premembrane [prM] and membrane [M] ), and envelope (E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (3) . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance.", "These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 (DENV-2). The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease.", "The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) .", "Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal (S/B) ratio, coefficient of variation (CV), and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig.", "A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected.", "After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) .", "1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine (DHP) voltage-gated Ca 2ϩ channel (VGCC) antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig.", "All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig. 1C) . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig.", "As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig. 2B) . A sharp decrease in JEV RNA levels was also detected (Fig. 2C) .", "A sharp decrease in JEV RNA levels was also detected (Fig. 2C) . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%.", "The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig.", "Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig. 2D) . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry.", "3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney (BHK-21) cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig.", "It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig. 3C) , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig.", "Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig. 4A) . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig.", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig. 4A) . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields.", "Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig.", "The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig. 4B and C). Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors.", "To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine (PAA) VGCC inhibitor (15) , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney (Vero) and human hepatocellular carcinoma (Huh-7) cells (Fig. 5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", "5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate (2-APB), which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum (ER) pool, respectively (16) (17) (18) (19) , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection.", "Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV.", "Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 (P20) showed robust resistance compared with the wild type (WT) (Fig. 6A ).", "6A ). When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified.", "Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine (Q)-to-arginine (R) switch at amino acid position 130 in transmembrane domain 3 (TMD3) of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone (Fig. 6B ).", "6B ). Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position (Fig. 6B) . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig.", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig. 4) , while YFV showed resistance to the drug (data not shown). To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses.", "To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig.", "Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig. 6C) , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine.", "6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine.", "In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs (Fig. 2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model.", "2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived).", "Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived). Manidipine treatment following JEV infection reduced the mortality rate to 20% (12 out of 15 animals survived) (Fig. 7A ).", "7A ). Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality.", "These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig.", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig. 7B) . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) .", "As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena (Fig. 7D) .", "7D) . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig.", "To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated.", "To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication.", "As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) .", "NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) .", "Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) .", "However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) .", "Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32.", "Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration (C max ) of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml (40) , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) .", "Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses.", "Cells and viruses. BHK-21, SH-SY5Y (human neuroblastoma), Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China.", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China. JEV replicon recombinant viral particles (RVPs) were generated as previously described (4, 5) . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells.", "ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay.", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities (2,500 to 12,500 cells per well) were infected with from 1.25 to 20 l RVPs (1 to 16 copies per well). The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) .", "The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) . Methyl-␤-cyclodextrin and dimethyl sulfoxide (DMSO) were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library.", "HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals (Houston, TX, USA). The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A .", "1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen.", "The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . The experimental timeline is depicted in Fig. 2A .", "The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) .", "To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) . Vero cells were infected with 20 l RVPs for 1 h (0 to 1 h). The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig.", "The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig. 3A) . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells.", "To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above (Fig. 3A) . Manidipine-resistant virus.", "3A) . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel.", "As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 (pMWJEAT), kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously (4) . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells.", "Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China).", "Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China). The mice were randomly divided into four groups (30 mice per group): a JEV-infected and vehicle (2% Tween 80 plus 5% DMSO in phosphate-buffered saline [PBS])-treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31.", "For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation.", "Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China)." ]

[ "Japanese encephalitis virus (JEV), an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed.", "In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile.", "Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca(2+) channel (VGCC) inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available.", "IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae.", "Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV). Most flaviviruses are arthropod borne and cause public health problems worldwide (1) .", "Most flaviviruses are arthropod borne and cause public health problems worldwide (1) . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus (TBEV), have decreased the rates of morbidity and mortality from infections caused by these viruses (2) ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini.", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both termini. The ORF encodes three structural proteins, including the capsid (C), membrane (premembrane [prM] and membrane [M] ), and envelope (E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (3) . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance.", "These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 (DENV-2). The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease.", "The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) .", "Recombinant viral particles (RVPs) with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening (HTS) assay (4, 5) . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l (16 copies/cell) RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal (S/B) ratio, coefficient of variation (CV), and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig.", "A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected.", "After three rounds of screening, five hits with a selective index (SI; which is equal to the 50% cytotoxic concentration [CC 50 [/50% inhibitory concentration [IC 50 ]) of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) .", "1B were similar to those previously published for diverse cell systems but determined using different toxicity assays (6) (7) (8) (9) (10) (11) (12) (13) . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine (DHP) voltage-gated Ca 2ϩ channel (VGCC) antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig.", "All five drugs exhibited a dose-dependent inhibition of JEV RVP infection (Fig. 1C) . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig.", "As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs (Fig. 2B) . A sharp decrease in JEV RNA levels was also detected (Fig. 2C) .", "A sharp decrease in JEV RNA levels was also detected (Fig. 2C) . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%.", "The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig.", "Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration (Fig. 2D) . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry.", "3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney (BHK-21) cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig.", "It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA (5, 14) (Fig. 3C) , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig.", "Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs (Fig. 4A) . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig.", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected (Fig. 4A) . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields.", "Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig.", "The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested ( Fig. 4B and C). Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors.", "To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine (PAA) VGCC inhibitor (15) , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney (Vero) and human hepatocellular carcinoma (Huh-7) cells (Fig. 5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", "5) , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate (2-APB), which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum (ER) pool, respectively (16) (17) (18) (19) , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection.", "Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV.", "Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 (P20) showed robust resistance compared with the wild type (WT) (Fig. 6A ).", "6A ). When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified.", "Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine (Q)-to-arginine (R) switch at amino acid position 130 in transmembrane domain 3 (TMD3) of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone (Fig. 6B ).", "6B ). Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position (Fig. 6B) . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig.", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above (Fig. 4) , while YFV showed resistance to the drug (data not shown). To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses.", "To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig.", "Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT (Fig. 6C) , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine.", "6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine.", "In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs (Fig. 2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model.", "2 and 4A) , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived).", "Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% (4 out of 15 animals survived). Manidipine treatment following JEV infection reduced the mortality rate to 20% (12 out of 15 animals survived) (Fig. 7A ).", "7A ). Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality.", "These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig.", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection (Fig. 7B) . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) .", "As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads (20, 21) . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena (Fig. 7D) .", "7D) . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry (15, 22) or replication (18) and even at the stage of budding (23) . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig.", "To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated.", "To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication.", "As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) .", "NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane (24) . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response (25) . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) .", "Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B (26) (27) (28) (29) (30) (31) (32) . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) .", "However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing (33, 34) . As manidipine was approved for use for the long-term treatment of hypertension (35, 36) , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) .", "Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development (37) (38) (39) . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32.", "Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration (C max ) of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml (40) , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) .", "Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 (HSV-1) and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease (41, 42) . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses.", "Cells and viruses. BHK-21, SH-SY5Y (human neuroblastoma), Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China.", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase (Rluc) were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China. JEV replicon recombinant viral particles (RVPs) were generated as previously described (4, 5) . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells.", "ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay.", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities (2,500 to 12,500 cells per well) were infected with from 1.25 to 20 l RVPs (1 to 16 copies per well). The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) .", "The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described (43) . Methyl-␤-cyclodextrin and dimethyl sulfoxide (DMSO) were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library.", "HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals (Houston, TX, USA). The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A .", "1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen.", "The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR (qRT-PCR), immunofluorescence assay (IFA), and plaque assay as previously reported (44) (45) (46) (47) . The experimental timeline is depicted in Fig. 2A .", "The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) .", "To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described (43) . Vero cells were infected with 20 l RVPs for 1 h (0 to 1 h). The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig.", "The test compounds were incubated with the cells for 1 h before infection (Ϫ1 to 0 h), during infection (0 to 1 h), and for 23 h postinfection (1 to 24 h) (Fig. 3A) . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells.", "To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above (Fig. 3A) . Manidipine-resistant virus.", "3A) . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel.", "As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 (pMWJEAT), kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously (4) . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells.", "Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China).", "Adult female BALB/c mice (age, 4 weeks) were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS (Wuhan, China). The mice were randomly divided into four groups (30 mice per group): a JEV-infected and vehicle (2% Tween 80 plus 5% DMSO in phosphate-buffered saline [PBS])-treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31.", "For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation.", "Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China)." ]

[ "Acute gastroenteritis is one of the main causes of mortality in humans and young animals. Domestic and mainly wild animals such as bats, small rodents and birds are highly diversified animals in relation to their habitats and ecological niches and are widely distributed geographically in environments of forest fragmentation in some areas of the Amazon, being considered important sources for viruses that affect humans and other animals. Due to the anthropical activities, these animals changed their natural habitat and adapted to urbanized environments, thus representing risks to human and animal health.", "Due to the anthropical activities, these animals changed their natural habitat and adapted to urbanized environments, thus representing risks to human and animal health. Although the knowledge of the global diversity of enteric viruses is scarce, there are reports demonstrating the detection of rotavirus in domestic animals and animals of productive systems, such as bovines and pigs. The present study investigated the prevalence of Rotavirus A in 648 fecal samples of different animal species from the northeastern mesoregion of the state of Pará, Brazil, which is characterized as an urbanized area with forest fragments.", "The present study investigated the prevalence of Rotavirus A in 648 fecal samples of different animal species from the northeastern mesoregion of the state of Pará, Brazil, which is characterized as an urbanized area with forest fragments. The fecal specimens were collected from October 2014 to April 2016 and subjected to a Qualitative Real-Time Polymerase Chain Reaction (RT-qPCR), using the NSP3 gene as a target. It was observed that 27.5% (178/648) of the samples presented positive results for RVA, with 178 samples distributed in birds (23.6%), canines (21.35%), chiropterans (17.98%), bovines (14.6%), horses (8.43%), small rodents (6.74%), pigs (3.93%) and felines (3.37%), demonstrating the circulation of RVA in domestic animals and suggesting that such proximity could cause transmissions between different species and the occurrence of rearrangements in the genome of RVA as already described in the literature, associated to the traces of environmental degradation in the studied areas.", "It was observed that 27.5% (178/648) of the samples presented positive results for RVA, with 178 samples distributed in birds (23.6%), canines (21.35%), chiropterans (17.98%), bovines (14.6%), horses (8.43%), small rodents (6.74%), pigs (3.93%) and felines (3.37%), demonstrating the circulation of RVA in domestic animals and suggesting that such proximity could cause transmissions between different species and the occurrence of rearrangements in the genome of RVA as already described in the literature, associated to the traces of environmental degradation in the studied areas. Text: Emerging and reemerging infectious diseases are increasing each year in several countries, with an impact both on human populations and on domestic and wild animals living in areas with considerable forest remnants [1] . Most of these diseases are of viral origin, suggesting the emergence and reemergence of viruses that are triggered by human activities that modify the environment [2] .", "Most of these diseases are of viral origin, suggesting the emergence and reemergence of viruses that are triggered by human activities that modify the environment [2] . The populations of wild animals that inhabit forest fragments are strategic groups for studies of public health and the transmission of zoonosis, given that they act as indicators in the assistance and intervention in the human populations, aiming at the prevention of outbreaks and epidemics [3] . Acute gastroenteritis can be caused by infection in the gastrointestinal tract, caused by different infectious or parasite agents [4] [5] [6] [7] .", "Acute gastroenteritis can be caused by infection in the gastrointestinal tract, caused by different infectious or parasite agents [4] [5] [6] [7] . They represent one of the main causes of mortality in humans, and in young animals, counting for about 25% of mortality [8] . Rotavirus is widely distributed in animals, which act as sources of rotavirus emergent strains, with these animals acting in the transmission between species and through reassortment leading to the emergence of new strains which have been reported in human infections [9] [10] [11] [12] .", "Rotavirus is widely distributed in animals, which act as sources of rotavirus emergent strains, with these animals acting in the transmission between species and through reassortment leading to the emergence of new strains which have been reported in human infections [9] [10] [11] [12] . The rotavirus (RV) belongs to the Reoviridae family and comprises nine species known as Rotavirus group A to I, with a recent proposal of the J species [13, 14] . Rotavirus A (RVA) is widespread worldwide and predominantly infects humans, bovines and other mammal species, as well as birds [15] .", "Rotavirus A (RVA) is widespread worldwide and predominantly infects humans, bovines and other mammal species, as well as birds [15] . They have a double-stranded ribonucleic acid (dsRNA) genome, divided into 11 segments coding for structural proteins (VP1-VP4, VP6 and VP7) and nonstructural (NSP1-NSP5/NSP6) proteins [16, 17] . There are records of a close relationship between Amazonian wildlife and human populations [18] , and this interaction is the effect of anthropogenic urbanization activities that result in the deforestation of forest areas, causing the degradation of previously isolated sites such as caves and small caves, a continuous and nature progressive process that has led not only to changes in wildlife habitats but also to a greater relationship with human populations in rural and urban environments, contributing to the occurrence and emergence of diseases different from what normally occurs in endemic regions [19] [20] [21] [22] .", "There are records of a close relationship between Amazonian wildlife and human populations [18] , and this interaction is the effect of anthropogenic urbanization activities that result in the deforestation of forest areas, causing the degradation of previously isolated sites such as caves and small caves, a continuous and nature progressive process that has led not only to changes in wildlife habitats but also to a greater relationship with human populations in rural and urban environments, contributing to the occurrence and emergence of diseases different from what normally occurs in endemic regions [19] [20] [21] [22] . Although the results of RVA have already been described globally [12, [23] [24] [25] [26] [27] [28] [29] [30] , in Brazil, the occurrence, diversity and role of rotavirus in these animals are still poorly studied, considering the large number of present species [4, [31] [32] [33] [34] . In the Brazilian Amazon, especially in the state of Pará, the city of Belém and Northeast metropolitan mesoregions are some of the areas with the highest indexes of environmental changes [35] , which are concentrated, along with the fact that the knowledge of the global diversity of enteric virus in animals is scarce [36] .", "In the Brazilian Amazon, especially in the state of Pará, the city of Belém and Northeast metropolitan mesoregions are some of the areas with the highest indexes of environmental changes [35] , which are concentrated, along with the fact that the knowledge of the global diversity of enteric virus in animals is scarce [36] . Therefore, it is important to monitor the health of domestic and wild animals in their natural habitat, especially in areas with anthropic alterations that have an interface with rural communities and enterprises, in order to investigate the occurrence of RVA in this population. These communities are ecologically complex, because they have multiple hosts and endless pathogens that may eventually circulate in contiguous urban centers, in addition to the fact that it should also be considered that there is still a lack of studies showing the significance of these viruses infecting this population, as in the context of epidemiological surveillance, these animals become important, since they can be considered as natural sources, with the possibility of transmission to humans [37] [38] [39] .", "These communities are ecologically complex, because they have multiple hosts and endless pathogens that may eventually circulate in contiguous urban centers, in addition to the fact that it should also be considered that there is still a lack of studies showing the significance of these viruses infecting this population, as in the context of epidemiological surveillance, these animals become important, since they can be considered as natural sources, with the possibility of transmission to humans [37] [38] [39] . The qualitative real-time polymerase chain reaction (qRT-PCR) used the NSP3 gene and the TaqMan probe from a highly conserved region of the rotavirus non-structural protein 3 (NSP3), which was previously used in samples from human origin and with low viral loads \n\nPrecipitation data were obtained from The Brazilian National Institute of Meteorology (Inmet for the years of capture in the Expedito Ribeiro Settlement (2014) and Açailândia (2015) of the Data Collection Platforms (PCDs) of Belém, located 50 km from Santa Bárbara do Pará, and Tracuateua, located 50 km from Peixe-Boi and 100 km from Viseu. Garmin GPSMap 64s Global Positioning System (GPS) coordinates were collected in the field.", "Garmin GPSMap 64s Global Positioning System (GPS) coordinates were collected in the field. The municipal boundaries were obtained on the website of the Brazilian Institute of Geography and Statistics (IBGE) ( and data on deforestation and land use were obtained from the PRODES [43] and TerraClass [44] Projects. PRODES has annual data in digital format since 2000 and TerraClass presents biannual data since 2004.", "PRODES has annual data in digital format since 2000 and TerraClass presents biannual data since 2004. The satellite image was generated using the sensor Sentinel 2 of the European Space Agency (ESA) ( web/sentinel/user-guides/sentinel-2-msi) with Open Access CC-BY License ( from the years of 2017 and 2018. All the data obtained was stored in a Geographic Database (BDG).", "All the data obtained was stored in a Geographic Database (BDG). The BDG was imported/ stored in a GIS for the editing of the graphic elements, establishment of topological relations between the elements and their respective attributes, spatial analysis and visualization of the result through thematic maps. For the present study, forest fragments of similar size, shape and Phyto physiology were chosen, considering an open peri urban matrix with similar soil use.", "For the present study, forest fragments of similar size, shape and Phyto physiology were chosen, considering an open peri urban matrix with similar soil use. The selected fragments were distributed within the mesoregions studied, and in each selected fragment fecal samples were randomly collected from domestic and wild animals [45] . Soil use classes were obtained from the TerraClass data mosaic from 2004 to 2016, because the study sites were in an area with a high cloud presence, which prevented observation (the area was not observed).", "Soil use classes were obtained from the TerraClass data mosaic from 2004 to 2016, because the study sites were in an area with a high cloud presence, which prevented observation (the area was not observed). The data processing, interpretation, visualization and spatial analysis were performed in ArcGIS software ( For the analysis of data related to the determination of the richness, composition and abundance of the fauna of the animals studied in the study area, considering the collection methods adopted and the species available in each city, each sample was considered as an independent sample. The richness of wild fauna and domestic animals was determined by the total number of species including all collection methods, and the similarity of species was made by the chi-square analysis between the samples of the different treatments with the aid of the EstimateS 8.0 software [46] .", "The richness of wild fauna and domestic animals was determined by the total number of species including all collection methods, and the similarity of species was made by the chi-square analysis between the samples of the different treatments with the aid of the EstimateS 8.0 software [46] . For the calculation of the Test T, the Statistica software was used, and the indices of infected animals in the two environments (forest fragment and peridomicile) were calculated for each treatment sampled by collection area, using the software Past 1.92. Aiming at comparing the values of the diversity indexes through the paired test, as well as the descriptive analysis of the anthropic effects [47] .", "Aiming at comparing the values of the diversity indexes through the paired test, as well as the descriptive analysis of the anthropic effects [47] . The data obtained for the occurrence of RVA and the questionnaires was inserted into a database for a descriptive analysis of the epidemiological profile of the animal population in the three forest ecosystems studied. In this analysis, descriptive statistical treatments were carried out, using customized \"row-columns\" type charts, referring to the data, in order to characterize the sample and quantify the results using absolute frequency values using the chi-square test and the Test T.\n\nPopulation study, collection of clinical specimens and laboratory methodology.", "In this analysis, descriptive statistical treatments were carried out, using customized \"row-columns\" type charts, referring to the data, in order to characterize the sample and quantify the results using absolute frequency values using the chi-square test and the Test T.\n\nPopulation study, collection of clinical specimens and laboratory methodology. The flying animals (wild birds and chiroptera) were captured using mist nets which were opened at dawn (4:00 a.m.) and closed in the morning (9:00 a.m.) and were inspected every one hour until the closing, with a sampling effort of 15 days. This research was approved by National All procedures with animals were performed by veterinarians, being birds and bats identified and released at the same capture site.", "This research was approved by National All procedures with animals were performed by veterinarians, being birds and bats identified and released at the same capture site. The fecal specimens were collected by stimulation of the rectal ampulla with the use of a \"Zaragatoa\", packed in cryogenic vials, identified, stored in liquid nitrogen, and later sent to the Laboratory. Wild animals (small non-flying mammals) were trapped within live-containment traps of the Tomahawk cage (size 45x16x16cm) and Sherman type aluminum (size 30x9x8cm).", "Wild animals (small non-flying mammals) were trapped within live-containment traps of the Tomahawk cage (size 45x16x16cm) and Sherman type aluminum (size 30x9x8cm). In each sample plot, 61 traps were distributed, 20 Shermans and 41 Tomahawks being baited with a mixture made with peanut paste, sardines, cod liver oil and corn meal, as well as fruit like banana, apple and pineapple. All the traps used were inspected daily in the morning, the baits being exchanged when necessary and later after the capture in bags of cloth and at least five specimens of each species were chosen for the collection of biological material.", "All the traps used were inspected daily in the morning, the baits being exchanged when necessary and later after the capture in bags of cloth and at least five specimens of each species were chosen for the collection of biological material. The wild animals were sedated with a combination of ketamine 20mg/kg and xylazine 2mg/kg intramuscularly and subsequently, euthanized with anesthetic overdose of 2% lidocaine in the foramen magnum, according to the recommendation of the National Council for the Control of Animal Experimentation (CONCEA). From October 2014 to April 2016, 1,282 fecal samples were collected from wild and domestic animals.", "From October 2014 to April 2016, 1,282 fecal samples were collected from wild and domestic animals. Amongthese, 648 (50.5%) samples were randomly selected for RVA research and handled in Level Three Biosafety Laboratory (NB3). The viral genome was extracted using the TRIZOL LS REAGENT protocol (INVITRO-GEN, USA/KIT QIAGEN), following the manufacturer's recommendations, with minor adaptation according to the protocol described in the supplemental data.", "The viral genome was extracted using the TRIZOL LS REAGENT protocol (INVITRO-GEN, USA/KIT QIAGEN), following the manufacturer's recommendations, with minor adaptation according to the protocol described in the supplemental data. The qRT-PCR was conducted according to Zeng et al. [40] for the detection of RVA using the NSP3 segment of RVA as the target gene sequence.", "[40] for the detection of RVA using the NSP3 segment of RVA as the target gene sequence. The assay was conducted in a mixture containing: RNAse-free H 2 O, TaqMan RT-PCR Mix (2x), TAqMan RT Enzyme Mix (40x), primers for the NSP3 gene, Primer NSP3 Forward (20mM), Primer NSP3 Reverse (20mM), probe NSP3 S (10nm), Template (RNA) 3μL, having a total reaction volume of 17μL and reverse transcription cycling of 50˚C, 30 minutes, denaturation of 95˚C, 10 minutes, annealing of 45 cycles of 95˚C, 15 seconds and extension of 60˚C, 1 minute. The analyzes were considered positive when presenting the cycle threshold (CT) � 40.", "The analyzes were considered positive when presenting the cycle threshold (CT) � 40. In order to guarantee a reliable test result, the measurements of contamination control were performed with the use of positive animal control (SA11 prototype) and a negative control (ultrapure water). All RVA-positive samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) according to Mijatovic et al [41] to genotyping low viral loads samples.", "All RVA-positive samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) according to Mijatovic et al [41] to genotyping low viral loads samples. First round was performed with consensus primers N-VP4F1/N-VP4R1 and the Nested-PCR was conducted with N-VP4F2/N-VP4R2 primers to amplify VP4 gene. Amplicons were purified and sequencing for VP4 gene using the same primers of Nested-PCR.", "Amplicons were purified and sequencing for VP4 gene using the same primers of Nested-PCR. The sequences were collected from an automated ABI Prism 3130xl DNA sequencer (Applied Biosystems). The sequence fragments were assembled and edited using the Geneious Bioinformatics software platform v.8.1.7.", "The sequence fragments were assembled and edited using the Geneious Bioinformatics software platform v.8.1.7. Posteriorly, the data were compared with othersequences from the National Center for Biotechnology Information GenBank database using BLAST alignment tool to elucidate the RVA genotype of the samples. From October 2014 to April 2016, a total of 648 fecal samples of wild and domestic animals belonging to three forest fragments areas were tested for the NSP3 gene by qualitative qPCR, and 178 (27.5%) were positive for RVA, distributed among the species: birds (23.6%), canines (21.35%), bats (17.98%), cattle (14.6%), horses (8.43%), small rodents (6.74%), swine (3.93%) and felines (3.37%).", "From October 2014 to April 2016, a total of 648 fecal samples of wild and domestic animals belonging to three forest fragments areas were tested for the NSP3 gene by qualitative qPCR, and 178 (27.5%) were positive for RVA, distributed among the species: birds (23.6%), canines (21.35%), bats (17.98%), cattle (14.6%), horses (8.43%), small rodents (6.74%), swine (3.93%) and felines (3.37%). The CT interval ranged from 28. 47 It was possible to detect viral strains in all genders of animals studied and in the harvesting period none of the animals showed signs of acute infection and / or diarrhea.", "47 It was possible to detect viral strains in all genders of animals studied and in the harvesting period none of the animals showed signs of acute infection and / or diarrhea. Rotavirus A (RVA) detected in the present study of wild and domestic animals belonging to the three areas of forest fragment, according to Fig 2. In relation to the evaluated bovines, only in the city of Viseu, these species were studied because they were created extensively.", "In relation to the evaluated bovines, only in the city of Viseu, these species were studied because they were created extensively. In addition, most of the animals were young with ages varying from 1 day to 8 yearsold, history of deficient vaccination, lack of technical assistance and raised in the form of subsistence. The animals showed no symptoms of diarrhea, only low weight performance and poor sanitary management status.", "The animals showed no symptoms of diarrhea, only low weight performance and poor sanitary management status. In relation to chiroptera, 32 (17.98%) positive samples for RVA were distributed among Carollia perspicillata species, with 12 (37.5%) being all adults, 9 (28.12%) Desmodus rotundus samples (4 young and 5 adults), 5 (15.6%) of Uroderma bilobata (15.62%), 3 (9.37%) of Artibeus lituratus and the species Artibeus Planirostus, Diaemus iyoug and Glossophagine with 1 (3.12%) each. These animals came from areas of forest fragments located near bovine and equine farms, in addition to inhabiting small chicken farms.", "These animals came from areas of forest fragments located near bovine and equine farms, in addition to inhabiting small chicken farms. Fig 3 shows the results obtained for all the species of animals investigated in the forest fragment as well as in the peridomicillus area. The anthropic variables were analyzed for the three cities studied, as well as the use of the soil within the range of the animals, obeying the domicile, the peridomicile and the forest fragment where the traps of small rodents, birds and various species of animals were captured (Fig 4 and Fig 5) .", "The anthropic variables were analyzed for the three cities studied, as well as the use of the soil within the range of the animals, obeying the domicile, the peridomicile and the forest fragment where the traps of small rodents, birds and various species of animals were captured (Fig 4 and Fig 5) . Considering the factors related to the anthropic activities in the three studied areas within the three cities of the present study, it was observed that the city of Santa Bárbara is the one that has a better area of preserved forest and the city of Viseu a smaller area. However, in the city of Santa Bárbara, a greater concentration of occupations was observed around the area of forest fragment.", "However, in the city of Santa Bárbara, a greater concentration of occupations was observed around the area of forest fragment. It was observed in this chosen area of the city, the presence of different families living in a rural settlement, surviving from the exploitation of forest resources and the creation of small animals for subsistence, such as poultry and fish farming, as well as family farming products. The breeding of animals in native pastures was only observed in the cities of Peixe Boi and Viseu.", "The breeding of animals in native pastures was only observed in the cities of Peixe Boi and Viseu. Extensive livestock farming was practiced with beef cattle, equines for work and small animals (swine and goats). In relation to the most preserved pasture area, the city of Peixe Boi had the largest area, according to the data shown in Fig 5, however, in the city of Viseu, a higher regeneration was observed in the pastures during the period of the study, with significant secondary vegetation.", "In relation to the most preserved pasture area, the city of Peixe Boi had the largest area, according to the data shown in Fig 5, however, in the city of Viseu, a higher regeneration was observed in the pastures during the period of the study, with significant secondary vegetation. When comparing the climates of the three areas it was observed that the predominant climate is megathermal and humid with average annual temperature around 27˚C. The months of October, November and December are the hottest, with temperatures between 32˚C and 34˚C and absolute maximums around 41˚C.", "The months of October, November and December are the hottest, with temperatures between 32˚C and 34˚C and absolute maximums around 41˚C. Annual rainfall is quite high, generally around 2,350 mm, but strongly concentrated from January to June (80%). From September to December, on the contrary, rainfall is rare, about 7%, with a short dry season, of moderate water deficit in those months.", "From September to December, on the contrary, rainfall is rare, about 7%, with a short dry season, of moderate water deficit in those months. The relative humidity of the average air oscillates around 85%, as shown in Fig 6 [48] . The description of the accumulated precipitation in the year of capture of the fecal specimens compared to the Climatological Normals (CLINO) for the period from 1961-1990 of the PCDs closest to the locations of the Expedito Ribeiro / Santa Bárbara settlement (Belém PCD), Vila Ananim / Peixe-Boi and Açaiteua / Viseu (Tracauateua PCD) show the frequency of rainfall in the regions, which facilitates the renewal of the pastures and the regeneration of the impacted forests, being an important indicator of the reduction of the damages caused by deforestation in the region.", "The description of the accumulated precipitation in the year of capture of the fecal specimens compared to the Climatological Normals (CLINO) for the period from 1961-1990 of the PCDs closest to the locations of the Expedito Ribeiro / Santa Bárbara settlement (Belém PCD), Vila Ananim / Peixe-Boi and Açaiteua / Viseu (Tracauateua PCD) show the frequency of rainfall in the regions, which facilitates the renewal of the pastures and the regeneration of the impacted forests, being an important indicator of the reduction of the damages caused by deforestation in the region. The average deforestation index in the three study areas was calculated from data obtained from INPE information systems. It was observed that in the years of 2013 to 2014 there were no changes in these regions; in the period from 2014 to 2015 about 4.1% of the city of Viseu was changed and 1.6% of the city of Peixe Boi.", "It was observed that in the years of 2013 to 2014 there were no changes in these regions; in the period from 2014 to 2015 about 4.1% of the city of Viseu was changed and 1.6% of the city of Peixe Boi. In relation to the period of 2016, great changes were observed in Peixe Boi (79%) and in the city of Viseu (70%), thus demonstrating that changes in the natural ecosystem may be associated with the frequencies for RVA in the studied areas, according to Fig 7. When assessing the infected animals in relation to the uninfected animals in both the forest fragment and the peridomicile, considering as animals of the forest fragment the birds, the chiroptera and the small rodents and as animals of the peridomicile the canines, bovines, pigs, felines and horses, a percentage of 37.07% infected peri domestic animals (86/232) and 22.12% infected forest fragment animals (92/416) were obtained.", "When assessing the infected animals in relation to the uninfected animals in both the forest fragment and the peridomicile, considering as animals of the forest fragment the birds, the chiroptera and the small rodents and as animals of the peridomicile the canines, bovines, pigs, felines and horses, a percentage of 37.07% infected peri domestic animals (86/232) and 22.12% infected forest fragment animals (92/416) were obtained. Applying the selected statistical analysis, a Pearson x2 Chi-square value was obtained: 16.7159, df = 1 and p <0.001, meaning that the hypothesis was corroborated, that is, the greater the degradation of the environment, the more likely it will be the search for food by wild animals in adjacent areas, or in the edge of the forest or even in the peri domiciliary region. In this sense, the possibility of contagion with other species of animals, even humans, should be considered because of the capacity of the rotavirus to be transmitted via the fecal / oral route or through direct contact with the environment.", "In this sense, the possibility of contagion with other species of animals, even humans, should be considered because of the capacity of the rotavirus to be transmitted via the fecal / oral route or through direct contact with the environment. It is important to point out that the animals detected in this study are important sources of viral strains. A total of 80 stool samples were selected, reextracted and analyzed using PCR for the VP4 gene.", "A total of 80 stool samples were selected, reextracted and analyzed using PCR for the VP4 gene. Eight strains (10%) were positive for VP4 gene, being 2 strains bellowed to P [6] genotype and 6 to P[4]-type, according to \n\nIn the present study, RVA was detected circulating in 27.5% of the animals; 36% in domestic animals and 64% in wild animals, providing a unique dataset with qRT-PCR detecting a low viral load of RVA in different species, which further correlates with the deforestation index. These data are important because there is a lack of tests for RVA diagnosis in animals, since the current methods of RVA detection does not always detect in these populations [8] .", "These data are important because there is a lack of tests for RVA diagnosis in animals, since the current methods of RVA detection does not always detect in these populations [8] . With the advent of real-time PCR (qPCR), there was an exponential growth, compared to conventional PCR essays, since its superior accuracy, sensitivity and specificity is remarkable, and it is Rotavirus A in wild and domestic animals possible to detect RVA in a variety of animal species using NSP3 gene [49] . The sensitivity of RT-qPCR significantly improved the rate of RVA detection in clinical samples from animals and in this context, the present study proposed an interesting study metrics using virus spreading in the wild animals which inhabit forest fragments to indicate human population interventions, with the goal of preventing the virus outbreaks leveraged on the unique geographic characteristics of Brazil and its large number of species in Amazon.", "The sensitivity of RT-qPCR significantly improved the rate of RVA detection in clinical samples from animals and in this context, the present study proposed an interesting study metrics using virus spreading in the wild animals which inhabit forest fragments to indicate human population interventions, with the goal of preventing the virus outbreaks leveraged on the unique geographic characteristics of Brazil and its large number of species in Amazon. Currently, no data have been described in the literature regarding the RVA detection using real-time qPCR technique in a wide variety of wild animal species. However, a study by Soltan et al.", "However, a study by Soltan et al. [50] conducted with horses and cattle detected RVA by RT-PCR, commercial RT-PCR and RT-qPCR in 36.7%, 51.4% and 56.9% respectively, differently from the present study that showed higher positivity for chiropterans (17.98%), canines (21.35%), birds (23.6%) and cattle (14.6%). The first description of RVA in chiroptera was recorded in feces of Eidolon helvum caught in Vihiga, Kenya [51] .", "The first description of RVA in chiroptera was recorded in feces of Eidolon helvum caught in Vihiga, Kenya [51] . Afterwards, several strains of RVA were detected by different molecular techniques involving chiroptera, in several countries, including Kenya (E. helvum), China (Rhinolophus hipposideros and Aselliscus stoliczkanus), France (Myotis mystacinus), Cameroon (E. helvum) and Brazil [31, [51] [52] [53] [54] [55] . The present study shows the occurrence of RVA in 17.98% of the chiroptera, being among the species Carollia perspicillata (37.5%), Desmodus rotundus (28.12%), Uroderma bilobata (15.6%), Artibeus lituratus (9.37%), Artibeus Planirostus (3.12%), Diaemus iyoug and Glossophagine (3.12%).", "The present study shows the occurrence of RVA in 17.98% of the chiroptera, being among the species Carollia perspicillata (37.5%), Desmodus rotundus (28.12%), Uroderma bilobata (15.6%), Artibeus lituratus (9.37%), Artibeus Planirostus (3.12%), Diaemus iyoug and Glossophagine (3.12%). Barquez et al. [56] reported that Desmodus rotundus is one of the three hematophagous species of the Phyllostomidae family, found throughout South America, Central America and Mexico.", "[56] reported that Desmodus rotundus is one of the three hematophagous species of the Phyllostomidae family, found throughout South America, Central America and Mexico. Of the positive chiroptera for RVA in the present study, a prevalence of 28.12% was of Desmodus rotundus. This species feeds on birds, can feed on mammals, mostly medium or large, facilitating the dissemination of viral spores among the community within the habitat, as observed in the present study.", "This species feeds on birds, can feed on mammals, mostly medium or large, facilitating the dissemination of viral spores among the community within the habitat, as observed in the present study. These findings show the importance of epidemiological data on the studied species due to the lack of studies involving species of neotropical chiroptera, and it is not possible to establish comparative parameters for these animals. Regarding the circulation of RVA in canines and birds, the prevalence was 53% and 29%, respectively.", "Regarding the circulation of RVA in canines and birds, the prevalence was 53% and 29%, respectively. Although in the Amazon region there are records of RVA, RVD, RVF and RVG that infect birds [57] [58] and RVD in migratory birds [59] , all were detected by RT-PCR assays differently from the present study which detected the RVA by RT-qPCR involving a variety of animal species. On the other hand, the prevalence in felines (16%) and pigs (22%) was lower, probably because there are few animals of these species in the region, as well as few creations.", "On the other hand, the prevalence in felines (16%) and pigs (22%) was lower, probably because there are few animals of these species in the region, as well as few creations. The study detected the presence of RVA in different species of animals both in areas near the home and in areas located in fragments of forest, characterized as forest remnants, since they were located in cities that suffered high environmental impacts due to vegetal extractivism, pasture formation for cattle breeding, exploitation of natural resources, and direct reflexes on the habitats of wild animals that can serve as virus sources, thus facilitating the dispersion of RVA among communities of coexisting animals. It is worth emphasizing that these animals have a greater contact with the human populations of the studied areas since they cohabit with the humans in the region, besides having a high flow of movement between the forest extracts and environments chosen for the present study.", "It is worth emphasizing that these animals have a greater contact with the human populations of the studied areas since they cohabit with the humans in the region, besides having a high flow of movement between the forest extracts and environments chosen for the present study. However, it is noteworthy that only in the communities of Santa Bárbara and Viseu were collected fecal specimens from asymptomatic humans for diarrhea and tested for RVA, but all were negative. It is notorious yet, the existence of different levels of degradation in the studied environments, considering the presented data.", "It is notorious yet, the existence of different levels of degradation in the studied environments, considering the presented data. The fragmentation of the forest generates many consequences on the Amazonian biota, being able to alter the diversity and the composition of the animal communities in the fragments and even to interfere in the ecological processes, without considering that the fragments of forest in the Amazon are influenced by the climate, possibly facilitating the dispersion of pathogens by the environment, since the wild animals detected in the present study are asymptomatic and have low viral load for RVA. The occurrence of RVA in this population of animals may explain the possibility of dispersion of viral strains, since there is a proximity to the human population, besides the biological characteristics of these species that may represent important sources for gastroenteric viruses, along with the fact that all animals were asymptomatic for diarrhea.", "The occurrence of RVA in this population of animals may explain the possibility of dispersion of viral strains, since there is a proximity to the human population, besides the biological characteristics of these species that may represent important sources for gastroenteric viruses, along with the fact that all animals were asymptomatic for diarrhea. Wild birds have unlimited flight capacity, were captured in an interface region between the peridomicile and forest fragments and it is believed that this region has not been influenced by anthropic activities such as those observed in the area of the present study. On the other hand, the breeding method for poultry and canines close to homes and the forest ecosystem, as they are created in the communities surveyed, probably facilitates direct contact with possible sources of contamination, since in the areas the use of septic tanks is deficient and sometimes non-existent, which may facilitate or even increase the risk of viral dispersion throughout the environment.", "On the other hand, the breeding method for poultry and canines close to homes and the forest ecosystem, as they are created in the communities surveyed, probably facilitates direct contact with possible sources of contamination, since in the areas the use of septic tanks is deficient and sometimes non-existent, which may facilitate or even increase the risk of viral dispersion throughout the environment. The high rates of increase and the analysis of land use in the researched areas may be important indicators of how these animals interact, since with deforestation, the populations of wild animals seek refuge in nearby communities facilitating the dispersal of infectious agents and the possible occurrence of carrier animals by direct contact or contamination of the local environment. To our knowledge, this is the first study in which a real-time PCR assay was applied for the detection of RVA involving a wide variety of domestic and wild animals, facilitating practical utility in epidemiological and molecular studies and assisting in a perspective in the elaboration of sanitary control and monitoring, preventing possible outbreaks in the studied communities.", "To our knowledge, this is the first study in which a real-time PCR assay was applied for the detection of RVA involving a wide variety of domestic and wild animals, facilitating practical utility in epidemiological and molecular studies and assisting in a perspective in the elaboration of sanitary control and monitoring, preventing possible outbreaks in the studied communities. The detection of positive animals was useful to monitor the infection of the agent in the animal population and to provide an early warning signal to predict an impending epidemic and a favorable risk for the human population, given the evidence of RVA circulation in the different forest fragments. In addition, the RT-qPCR assay may be a useful alternative for the differential diagnosis of RV in possible coexisting mixed infections clinically indistinguishable such as those caused by other viral strains that cause gastroenteritis such as: astrovirus, coronavirus, picobirnavirus, calicivirus, among others as observed in the studies of Jing et al.", "In addition, the RT-qPCR assay may be a useful alternative for the differential diagnosis of RV in possible coexisting mixed infections clinically indistinguishable such as those caused by other viral strains that cause gastroenteritis such as: astrovirus, coronavirus, picobirnavirus, calicivirus, among others as observed in the studies of Jing et al. [60] and Waruhiu et al. [61] .", "[60] and Waruhiu et al. [61] . Diarrhea associated with RVA infections in pigs is an important cause of increased mortality and economic losses in Europe.", "Diarrhea associated with RVA infections in pigs is an important cause of increased mortality and economic losses in Europe. The most prevalent genotypes isolated from feces of Belgian diarrheal and non-diarrheal piglets in 2012 [62] demonstrate a wide range of combinations of genotypes G / P including; G3P [6] , G4P [6] , G5P [6] , G4P [7] , G5P [7] , G9P [7] , G9P [13] and G9P [23] . On the other hand, in the present study it was possible to detect only P [6] genotype, since majority of samples was asymptomatic for diarrhea.", "On the other hand, in the present study it was possible to detect only P [6] genotype, since majority of samples was asymptomatic for diarrhea. Finding shows that different P genotypes of RVA strains interact with distinct blood group histological antigens (HBGA, ABOH, Lewis) and sialic acids via VP4 providing insight into the regional prevalence and increased zoonotic potential of some RVA of origin swine [63] . The genotype P [6] was identified in piglets in Brazil [64] and in Italy and Japan resembling genotype P [6] human [65, 66] .", "The genotype P [6] was identified in piglets in Brazil [64] and in Italy and Japan resembling genotype P [6] human [65, 66] . In the population of animals studied the zoonotic transmission can be frequent, since the animals live in contact with humans and in precarious sanitary conditions. In Brazil, this genotype was described in animal and human populations in studies of Luchs et al.", "In Brazil, this genotype was described in animal and human populations in studies of Luchs et al. [32] ; Honma et al. [67] ; Araújo et al. [68] ; Mascarenhas et al. [69] and Lorenzetti et al.", "[68] ; Mascarenhas et al. [69] and Lorenzetti et al. [70] such studies corroborate the importance of continuing to monitor genotypes to verify if uncommon strains or new strains are emerging and can infect animal populations or inter-species transmissions.", "[70] such studies corroborate the importance of continuing to monitor genotypes to verify if uncommon strains or new strains are emerging and can infect animal populations or inter-species transmissions. Regarding the genotype P [4] , itwas most detected in our samples in bats, dogs, swine and feline. This genotype is not common in animals, being more detected in human and environmental samples in various parts of the world and included our region [71] .", "This genotype is not common in animals, being more detected in human and environmental samples in various parts of the world and included our region [71] . It is important to emphasize that the indicators of environmental contamination in Brazil are significant and contribute to the possibility of human-animal transmission [71] . Such data need further investigation in later work to better characterize the interspecies transmission, since the occurrence of enteric viruses in different matrices demonstrates the anthropogenic impact of the exposed population around and points to the potential risk of infection by the possible exposure of individuals susceptible.", "Such data need further investigation in later work to better characterize the interspecies transmission, since the occurrence of enteric viruses in different matrices demonstrates the anthropogenic impact of the exposed population around and points to the potential risk of infection by the possible exposure of individuals susceptible. Our findings may be useful for tracking fecal contamination in the environment using animals as possible sources thus minimizing the risk of infection by exposure to susceptible individuals, in this case different animal species or even human populations. RVA were detected in wild and domestic animals using a RT-qPCR assay that analyzed samples that had low viral load for RVA.", "RVA were detected in wild and domestic animals using a RT-qPCR assay that analyzed samples that had low viral load for RVA. Although the samples are asymptomatic for diarrhea, it is necessary to conduct strategies for the monitoring and control of the animals in the areas studied in the human population as well as in other species of animals, as well as the implementation of preventive measures aimed at future outbreaks in communities animals in the resident population in these impacted areas. Therefore, the present study is unprecedented in the region and in the country in relation to the research of RVA in wild animals.", "Therefore, the present study is unprecedented in the region and in the country in relation to the research of RVA in wild animals. It is noteworthy that, although the quality of the analyzed samples is characterized as low detectable viral load, the technique presented a good analytical response in the detection of the source animals for RVA, facilitating the selection of the samples for future genetic characterization tests." ]

[ "Acute gastroenteritis is one of the main causes of mortality in humans and young animals. Domestic and mainly wild animals such as bats, small rodents and birds are highly diversified animals in relation to their habitats and ecological niches and are widely distributed geographically in environments of forest fragmentation in some areas of the Amazon, being considered important sources for viruses that affect humans and other animals. Due to the anthropical activities, these animals changed their natural habitat and adapted to urbanized environments, thus representing risks to human and animal health.", "Due to the anthropical activities, these animals changed their natural habitat and adapted to urbanized environments, thus representing risks to human and animal health. Although the knowledge of the global diversity of enteric viruses is scarce, there are reports demonstrating the detection of rotavirus in domestic animals and animals of productive systems, such as bovines and pigs. The present study investigated the prevalence of Rotavirus A in 648 fecal samples of different animal species from the northeastern mesoregion of the state of Pará, Brazil, which is characterized as an urbanized area with forest fragments.", "The present study investigated the prevalence of Rotavirus A in 648 fecal samples of different animal species from the northeastern mesoregion of the state of Pará, Brazil, which is characterized as an urbanized area with forest fragments. The fecal specimens were collected from October 2014 to April 2016 and subjected to a Qualitative Real-Time Polymerase Chain Reaction (RT-qPCR), using the NSP3 gene as a target. It was observed that 27.5% (178/648) of the samples presented positive results for RVA, with 178 samples distributed in birds (23.6%), canines (21.35%), chiropterans (17.98%), bovines (14.6%), horses (8.43%), small rodents (6.74%), pigs (3.93%) and felines (3.37%), demonstrating the circulation of RVA in domestic animals and suggesting that such proximity could cause transmissions between different species and the occurrence of rearrangements in the genome of RVA as already described in the literature, associated to the traces of environmental degradation in the studied areas.", "It was observed that 27.5% (178/648) of the samples presented positive results for RVA, with 178 samples distributed in birds (23.6%), canines (21.35%), chiropterans (17.98%), bovines (14.6%), horses (8.43%), small rodents (6.74%), pigs (3.93%) and felines (3.37%), demonstrating the circulation of RVA in domestic animals and suggesting that such proximity could cause transmissions between different species and the occurrence of rearrangements in the genome of RVA as already described in the literature, associated to the traces of environmental degradation in the studied areas. Text: Emerging and reemerging infectious diseases are increasing each year in several countries, with an impact both on human populations and on domestic and wild animals living in areas with considerable forest remnants [1] . Most of these diseases are of viral origin, suggesting the emergence and reemergence of viruses that are triggered by human activities that modify the environment [2] .", "Most of these diseases are of viral origin, suggesting the emergence and reemergence of viruses that are triggered by human activities that modify the environment [2] . The populations of wild animals that inhabit forest fragments are strategic groups for studies of public health and the transmission of zoonosis, given that they act as indicators in the assistance and intervention in the human populations, aiming at the prevention of outbreaks and epidemics [3] . Acute gastroenteritis can be caused by infection in the gastrointestinal tract, caused by different infectious or parasite agents [4] [5] [6] [7] .", "Acute gastroenteritis can be caused by infection in the gastrointestinal tract, caused by different infectious or parasite agents [4] [5] [6] [7] . They represent one of the main causes of mortality in humans, and in young animals, counting for about 25% of mortality [8] . Rotavirus is widely distributed in animals, which act as sources of rotavirus emergent strains, with these animals acting in the transmission between species and through reassortment leading to the emergence of new strains which have been reported in human infections [9] [10] [11] [12] .", "Rotavirus is widely distributed in animals, which act as sources of rotavirus emergent strains, with these animals acting in the transmission between species and through reassortment leading to the emergence of new strains which have been reported in human infections [9] [10] [11] [12] . The rotavirus (RV) belongs to the Reoviridae family and comprises nine species known as Rotavirus group A to I, with a recent proposal of the J species [13, 14] . Rotavirus A (RVA) is widespread worldwide and predominantly infects humans, bovines and other mammal species, as well as birds [15] .", "Rotavirus A (RVA) is widespread worldwide and predominantly infects humans, bovines and other mammal species, as well as birds [15] . They have a double-stranded ribonucleic acid (dsRNA) genome, divided into 11 segments coding for structural proteins (VP1-VP4, VP6 and VP7) and nonstructural (NSP1-NSP5/NSP6) proteins [16, 17] . There are records of a close relationship between Amazonian wildlife and human populations [18] , and this interaction is the effect of anthropogenic urbanization activities that result in the deforestation of forest areas, causing the degradation of previously isolated sites such as caves and small caves, a continuous and nature progressive process that has led not only to changes in wildlife habitats but also to a greater relationship with human populations in rural and urban environments, contributing to the occurrence and emergence of diseases different from what normally occurs in endemic regions [19] [20] [21] [22] .", "There are records of a close relationship between Amazonian wildlife and human populations [18] , and this interaction is the effect of anthropogenic urbanization activities that result in the deforestation of forest areas, causing the degradation of previously isolated sites such as caves and small caves, a continuous and nature progressive process that has led not only to changes in wildlife habitats but also to a greater relationship with human populations in rural and urban environments, contributing to the occurrence and emergence of diseases different from what normally occurs in endemic regions [19] [20] [21] [22] . Although the results of RVA have already been described globally [12, [23] [24] [25] [26] [27] [28] [29] [30] , in Brazil, the occurrence, diversity and role of rotavirus in these animals are still poorly studied, considering the large number of present species [4, [31] [32] [33] [34] . In the Brazilian Amazon, especially in the state of Pará, the city of Belém and Northeast metropolitan mesoregions are some of the areas with the highest indexes of environmental changes [35] , which are concentrated, along with the fact that the knowledge of the global diversity of enteric virus in animals is scarce [36] .", "In the Brazilian Amazon, especially in the state of Pará, the city of Belém and Northeast metropolitan mesoregions are some of the areas with the highest indexes of environmental changes [35] , which are concentrated, along with the fact that the knowledge of the global diversity of enteric virus in animals is scarce [36] . Therefore, it is important to monitor the health of domestic and wild animals in their natural habitat, especially in areas with anthropic alterations that have an interface with rural communities and enterprises, in order to investigate the occurrence of RVA in this population. These communities are ecologically complex, because they have multiple hosts and endless pathogens that may eventually circulate in contiguous urban centers, in addition to the fact that it should also be considered that there is still a lack of studies showing the significance of these viruses infecting this population, as in the context of epidemiological surveillance, these animals become important, since they can be considered as natural sources, with the possibility of transmission to humans [37] [38] [39] .", "These communities are ecologically complex, because they have multiple hosts and endless pathogens that may eventually circulate in contiguous urban centers, in addition to the fact that it should also be considered that there is still a lack of studies showing the significance of these viruses infecting this population, as in the context of epidemiological surveillance, these animals become important, since they can be considered as natural sources, with the possibility of transmission to humans [37] [38] [39] . The qualitative real-time polymerase chain reaction (qRT-PCR) used the NSP3 gene and the TaqMan probe from a highly conserved region of the rotavirus non-structural protein 3 (NSP3), which was previously used in samples from human origin and with low viral loads \n\nPrecipitation data were obtained from The Brazilian National Institute of Meteorology (Inmet for the years of capture in the Expedito Ribeiro Settlement (2014) and Açailândia (2015) of the Data Collection Platforms (PCDs) of Belém, located 50 km from Santa Bárbara do Pará, and Tracuateua, located 50 km from Peixe-Boi and 100 km from Viseu. Garmin GPSMap 64s Global Positioning System (GPS) coordinates were collected in the field.", "Garmin GPSMap 64s Global Positioning System (GPS) coordinates were collected in the field. The municipal boundaries were obtained on the website of the Brazilian Institute of Geography and Statistics (IBGE) ( and data on deforestation and land use were obtained from the PRODES [43] and TerraClass [44] Projects. PRODES has annual data in digital format since 2000 and TerraClass presents biannual data since 2004.", "PRODES has annual data in digital format since 2000 and TerraClass presents biannual data since 2004. The satellite image was generated using the sensor Sentinel 2 of the European Space Agency (ESA) ( web/sentinel/user-guides/sentinel-2-msi) with Open Access CC-BY License ( from the years of 2017 and 2018. All the data obtained was stored in a Geographic Database (BDG).", "All the data obtained was stored in a Geographic Database (BDG). The BDG was imported/ stored in a GIS for the editing of the graphic elements, establishment of topological relations between the elements and their respective attributes, spatial analysis and visualization of the result through thematic maps. For the present study, forest fragments of similar size, shape and Phyto physiology were chosen, considering an open peri urban matrix with similar soil use.", "For the present study, forest fragments of similar size, shape and Phyto physiology were chosen, considering an open peri urban matrix with similar soil use. The selected fragments were distributed within the mesoregions studied, and in each selected fragment fecal samples were randomly collected from domestic and wild animals [45] . Soil use classes were obtained from the TerraClass data mosaic from 2004 to 2016, because the study sites were in an area with a high cloud presence, which prevented observation (the area was not observed).", "Soil use classes were obtained from the TerraClass data mosaic from 2004 to 2016, because the study sites were in an area with a high cloud presence, which prevented observation (the area was not observed). The data processing, interpretation, visualization and spatial analysis were performed in ArcGIS software ( For the analysis of data related to the determination of the richness, composition and abundance of the fauna of the animals studied in the study area, considering the collection methods adopted and the species available in each city, each sample was considered as an independent sample. The richness of wild fauna and domestic animals was determined by the total number of species including all collection methods, and the similarity of species was made by the chi-square analysis between the samples of the different treatments with the aid of the EstimateS 8.0 software [46] .", "The richness of wild fauna and domestic animals was determined by the total number of species including all collection methods, and the similarity of species was made by the chi-square analysis between the samples of the different treatments with the aid of the EstimateS 8.0 software [46] . For the calculation of the Test T, the Statistica software was used, and the indices of infected animals in the two environments (forest fragment and peridomicile) were calculated for each treatment sampled by collection area, using the software Past 1.92. Aiming at comparing the values of the diversity indexes through the paired test, as well as the descriptive analysis of the anthropic effects [47] .", "Aiming at comparing the values of the diversity indexes through the paired test, as well as the descriptive analysis of the anthropic effects [47] . The data obtained for the occurrence of RVA and the questionnaires was inserted into a database for a descriptive analysis of the epidemiological profile of the animal population in the three forest ecosystems studied. In this analysis, descriptive statistical treatments were carried out, using customized \"row-columns\" type charts, referring to the data, in order to characterize the sample and quantify the results using absolute frequency values using the chi-square test and the Test T.\n\nPopulation study, collection of clinical specimens and laboratory methodology.", "In this analysis, descriptive statistical treatments were carried out, using customized \"row-columns\" type charts, referring to the data, in order to characterize the sample and quantify the results using absolute frequency values using the chi-square test and the Test T.\n\nPopulation study, collection of clinical specimens and laboratory methodology. The flying animals (wild birds and chiroptera) were captured using mist nets which were opened at dawn (4:00 a.m.) and closed in the morning (9:00 a.m.) and were inspected every one hour until the closing, with a sampling effort of 15 days. This research was approved by National All procedures with animals were performed by veterinarians, being birds and bats identified and released at the same capture site.", "This research was approved by National All procedures with animals were performed by veterinarians, being birds and bats identified and released at the same capture site. The fecal specimens were collected by stimulation of the rectal ampulla with the use of a \"Zaragatoa\", packed in cryogenic vials, identified, stored in liquid nitrogen, and later sent to the Laboratory. Wild animals (small non-flying mammals) were trapped within live-containment traps of the Tomahawk cage (size 45x16x16cm) and Sherman type aluminum (size 30x9x8cm).", "Wild animals (small non-flying mammals) were trapped within live-containment traps of the Tomahawk cage (size 45x16x16cm) and Sherman type aluminum (size 30x9x8cm). In each sample plot, 61 traps were distributed, 20 Shermans and 41 Tomahawks being baited with a mixture made with peanut paste, sardines, cod liver oil and corn meal, as well as fruit like banana, apple and pineapple. All the traps used were inspected daily in the morning, the baits being exchanged when necessary and later after the capture in bags of cloth and at least five specimens of each species were chosen for the collection of biological material.", "All the traps used were inspected daily in the morning, the baits being exchanged when necessary and later after the capture in bags of cloth and at least five specimens of each species were chosen for the collection of biological material. The wild animals were sedated with a combination of ketamine 20mg/kg and xylazine 2mg/kg intramuscularly and subsequently, euthanized with anesthetic overdose of 2% lidocaine in the foramen magnum, according to the recommendation of the National Council for the Control of Animal Experimentation (CONCEA). From October 2014 to April 2016, 1,282 fecal samples were collected from wild and domestic animals.", "From October 2014 to April 2016, 1,282 fecal samples were collected from wild and domestic animals. Amongthese, 648 (50.5%) samples were randomly selected for RVA research and handled in Level Three Biosafety Laboratory (NB3). The viral genome was extracted using the TRIZOL LS REAGENT protocol (INVITRO-GEN, USA/KIT QIAGEN), following the manufacturer's recommendations, with minor adaptation according to the protocol described in the supplemental data.", "The viral genome was extracted using the TRIZOL LS REAGENT protocol (INVITRO-GEN, USA/KIT QIAGEN), following the manufacturer's recommendations, with minor adaptation according to the protocol described in the supplemental data. The qRT-PCR was conducted according to Zeng et al. [40] for the detection of RVA using the NSP3 segment of RVA as the target gene sequence.", "[40] for the detection of RVA using the NSP3 segment of RVA as the target gene sequence. The assay was conducted in a mixture containing: RNAse-free H 2 O, TaqMan RT-PCR Mix (2x), TAqMan RT Enzyme Mix (40x), primers for the NSP3 gene, Primer NSP3 Forward (20mM), Primer NSP3 Reverse (20mM), probe NSP3 S (10nm), Template (RNA) 3μL, having a total reaction volume of 17μL and reverse transcription cycling of 50˚C, 30 minutes, denaturation of 95˚C, 10 minutes, annealing of 45 cycles of 95˚C, 15 seconds and extension of 60˚C, 1 minute. The analyzes were considered positive when presenting the cycle threshold (CT) � 40.", "The analyzes were considered positive when presenting the cycle threshold (CT) � 40. In order to guarantee a reliable test result, the measurements of contamination control were performed with the use of positive animal control (SA11 prototype) and a negative control (ultrapure water). All RVA-positive samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) according to Mijatovic et al [41] to genotyping low viral loads samples.", "All RVA-positive samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) according to Mijatovic et al [41] to genotyping low viral loads samples. First round was performed with consensus primers N-VP4F1/N-VP4R1 and the Nested-PCR was conducted with N-VP4F2/N-VP4R2 primers to amplify VP4 gene. Amplicons were purified and sequencing for VP4 gene using the same primers of Nested-PCR.", "Amplicons were purified and sequencing for VP4 gene using the same primers of Nested-PCR. The sequences were collected from an automated ABI Prism 3130xl DNA sequencer (Applied Biosystems). The sequence fragments were assembled and edited using the Geneious Bioinformatics software platform v.8.1.7.", "The sequence fragments were assembled and edited using the Geneious Bioinformatics software platform v.8.1.7. Posteriorly, the data were compared with othersequences from the National Center for Biotechnology Information GenBank database using BLAST alignment tool to elucidate the RVA genotype of the samples. From October 2014 to April 2016, a total of 648 fecal samples of wild and domestic animals belonging to three forest fragments areas were tested for the NSP3 gene by qualitative qPCR, and 178 (27.5%) were positive for RVA, distributed among the species: birds (23.6%), canines (21.35%), bats (17.98%), cattle (14.6%), horses (8.43%), small rodents (6.74%), swine (3.93%) and felines (3.37%).", "From October 2014 to April 2016, a total of 648 fecal samples of wild and domestic animals belonging to three forest fragments areas were tested for the NSP3 gene by qualitative qPCR, and 178 (27.5%) were positive for RVA, distributed among the species: birds (23.6%), canines (21.35%), bats (17.98%), cattle (14.6%), horses (8.43%), small rodents (6.74%), swine (3.93%) and felines (3.37%). The CT interval ranged from 28. 47 It was possible to detect viral strains in all genders of animals studied and in the harvesting period none of the animals showed signs of acute infection and / or diarrhea.", "47 It was possible to detect viral strains in all genders of animals studied and in the harvesting period none of the animals showed signs of acute infection and / or diarrhea. Rotavirus A (RVA) detected in the present study of wild and domestic animals belonging to the three areas of forest fragment, according to Fig 2. In relation to the evaluated bovines, only in the city of Viseu, these species were studied because they were created extensively.", "In relation to the evaluated bovines, only in the city of Viseu, these species were studied because they were created extensively. In addition, most of the animals were young with ages varying from 1 day to 8 yearsold, history of deficient vaccination, lack of technical assistance and raised in the form of subsistence. The animals showed no symptoms of diarrhea, only low weight performance and poor sanitary management status.", "The animals showed no symptoms of diarrhea, only low weight performance and poor sanitary management status. In relation to chiroptera, 32 (17.98%) positive samples for RVA were distributed among Carollia perspicillata species, with 12 (37.5%) being all adults, 9 (28.12%) Desmodus rotundus samples (4 young and 5 adults), 5 (15.6%) of Uroderma bilobata (15.62%), 3 (9.37%) of Artibeus lituratus and the species Artibeus Planirostus, Diaemus iyoug and Glossophagine with 1 (3.12%) each. These animals came from areas of forest fragments located near bovine and equine farms, in addition to inhabiting small chicken farms.", "These animals came from areas of forest fragments located near bovine and equine farms, in addition to inhabiting small chicken farms. Fig 3 shows the results obtained for all the species of animals investigated in the forest fragment as well as in the peridomicillus area. The anthropic variables were analyzed for the three cities studied, as well as the use of the soil within the range of the animals, obeying the domicile, the peridomicile and the forest fragment where the traps of small rodents, birds and various species of animals were captured (Fig 4 and Fig 5) .", "The anthropic variables were analyzed for the three cities studied, as well as the use of the soil within the range of the animals, obeying the domicile, the peridomicile and the forest fragment where the traps of small rodents, birds and various species of animals were captured (Fig 4 and Fig 5) . Considering the factors related to the anthropic activities in the three studied areas within the three cities of the present study, it was observed that the city of Santa Bárbara is the one that has a better area of preserved forest and the city of Viseu a smaller area. However, in the city of Santa Bárbara, a greater concentration of occupations was observed around the area of forest fragment.", "However, in the city of Santa Bárbara, a greater concentration of occupations was observed around the area of forest fragment. It was observed in this chosen area of the city, the presence of different families living in a rural settlement, surviving from the exploitation of forest resources and the creation of small animals for subsistence, such as poultry and fish farming, as well as family farming products. The breeding of animals in native pastures was only observed in the cities of Peixe Boi and Viseu.", "The breeding of animals in native pastures was only observed in the cities of Peixe Boi and Viseu. Extensive livestock farming was practiced with beef cattle, equines for work and small animals (swine and goats). In relation to the most preserved pasture area, the city of Peixe Boi had the largest area, according to the data shown in Fig 5, however, in the city of Viseu, a higher regeneration was observed in the pastures during the period of the study, with significant secondary vegetation.", "In relation to the most preserved pasture area, the city of Peixe Boi had the largest area, according to the data shown in Fig 5, however, in the city of Viseu, a higher regeneration was observed in the pastures during the period of the study, with significant secondary vegetation. When comparing the climates of the three areas it was observed that the predominant climate is megathermal and humid with average annual temperature around 27˚C. The months of October, November and December are the hottest, with temperatures between 32˚C and 34˚C and absolute maximums around 41˚C.", "The months of October, November and December are the hottest, with temperatures between 32˚C and 34˚C and absolute maximums around 41˚C. Annual rainfall is quite high, generally around 2,350 mm, but strongly concentrated from January to June (80%). From September to December, on the contrary, rainfall is rare, about 7%, with a short dry season, of moderate water deficit in those months.", "From September to December, on the contrary, rainfall is rare, about 7%, with a short dry season, of moderate water deficit in those months. The relative humidity of the average air oscillates around 85%, as shown in Fig 6 [48] . The description of the accumulated precipitation in the year of capture of the fecal specimens compared to the Climatological Normals (CLINO) for the period from 1961-1990 of the PCDs closest to the locations of the Expedito Ribeiro / Santa Bárbara settlement (Belém PCD), Vila Ananim / Peixe-Boi and Açaiteua / Viseu (Tracauateua PCD) show the frequency of rainfall in the regions, which facilitates the renewal of the pastures and the regeneration of the impacted forests, being an important indicator of the reduction of the damages caused by deforestation in the region.", "The description of the accumulated precipitation in the year of capture of the fecal specimens compared to the Climatological Normals (CLINO) for the period from 1961-1990 of the PCDs closest to the locations of the Expedito Ribeiro / Santa Bárbara settlement (Belém PCD), Vila Ananim / Peixe-Boi and Açaiteua / Viseu (Tracauateua PCD) show the frequency of rainfall in the regions, which facilitates the renewal of the pastures and the regeneration of the impacted forests, being an important indicator of the reduction of the damages caused by deforestation in the region. The average deforestation index in the three study areas was calculated from data obtained from INPE information systems. It was observed that in the years of 2013 to 2014 there were no changes in these regions; in the period from 2014 to 2015 about 4.1% of the city of Viseu was changed and 1.6% of the city of Peixe Boi.", "It was observed that in the years of 2013 to 2014 there were no changes in these regions; in the period from 2014 to 2015 about 4.1% of the city of Viseu was changed and 1.6% of the city of Peixe Boi. In relation to the period of 2016, great changes were observed in Peixe Boi (79%) and in the city of Viseu (70%), thus demonstrating that changes in the natural ecosystem may be associated with the frequencies for RVA in the studied areas, according to Fig 7. When assessing the infected animals in relation to the uninfected animals in both the forest fragment and the peridomicile, considering as animals of the forest fragment the birds, the chiroptera and the small rodents and as animals of the peridomicile the canines, bovines, pigs, felines and horses, a percentage of 37.07% infected peri domestic animals (86/232) and 22.12% infected forest fragment animals (92/416) were obtained.", "When assessing the infected animals in relation to the uninfected animals in both the forest fragment and the peridomicile, considering as animals of the forest fragment the birds, the chiroptera and the small rodents and as animals of the peridomicile the canines, bovines, pigs, felines and horses, a percentage of 37.07% infected peri domestic animals (86/232) and 22.12% infected forest fragment animals (92/416) were obtained. Applying the selected statistical analysis, a Pearson x2 Chi-square value was obtained: 16.7159, df = 1 and p <0.001, meaning that the hypothesis was corroborated, that is, the greater the degradation of the environment, the more likely it will be the search for food by wild animals in adjacent areas, or in the edge of the forest or even in the peri domiciliary region. In this sense, the possibility of contagion with other species of animals, even humans, should be considered because of the capacity of the rotavirus to be transmitted via the fecal / oral route or through direct contact with the environment.", "In this sense, the possibility of contagion with other species of animals, even humans, should be considered because of the capacity of the rotavirus to be transmitted via the fecal / oral route or through direct contact with the environment. It is important to point out that the animals detected in this study are important sources of viral strains. A total of 80 stool samples were selected, reextracted and analyzed using PCR for the VP4 gene.", "A total of 80 stool samples were selected, reextracted and analyzed using PCR for the VP4 gene. Eight strains (10%) were positive for VP4 gene, being 2 strains bellowed to P [6] genotype and 6 to P[4]-type, according to \n\nIn the present study, RVA was detected circulating in 27.5% of the animals; 36% in domestic animals and 64% in wild animals, providing a unique dataset with qRT-PCR detecting a low viral load of RVA in different species, which further correlates with the deforestation index. These data are important because there is a lack of tests for RVA diagnosis in animals, since the current methods of RVA detection does not always detect in these populations [8] .", "These data are important because there is a lack of tests for RVA diagnosis in animals, since the current methods of RVA detection does not always detect in these populations [8] . With the advent of real-time PCR (qPCR), there was an exponential growth, compared to conventional PCR essays, since its superior accuracy, sensitivity and specificity is remarkable, and it is Rotavirus A in wild and domestic animals possible to detect RVA in a variety of animal species using NSP3 gene [49] . The sensitivity of RT-qPCR significantly improved the rate of RVA detection in clinical samples from animals and in this context, the present study proposed an interesting study metrics using virus spreading in the wild animals which inhabit forest fragments to indicate human population interventions, with the goal of preventing the virus outbreaks leveraged on the unique geographic characteristics of Brazil and its large number of species in Amazon.", "The sensitivity of RT-qPCR significantly improved the rate of RVA detection in clinical samples from animals and in this context, the present study proposed an interesting study metrics using virus spreading in the wild animals which inhabit forest fragments to indicate human population interventions, with the goal of preventing the virus outbreaks leveraged on the unique geographic characteristics of Brazil and its large number of species in Amazon. Currently, no data have been described in the literature regarding the RVA detection using real-time qPCR technique in a wide variety of wild animal species. However, a study by Soltan et al.", "However, a study by Soltan et al. [50] conducted with horses and cattle detected RVA by RT-PCR, commercial RT-PCR and RT-qPCR in 36.7%, 51.4% and 56.9% respectively, differently from the present study that showed higher positivity for chiropterans (17.98%), canines (21.35%), birds (23.6%) and cattle (14.6%). The first description of RVA in chiroptera was recorded in feces of Eidolon helvum caught in Vihiga, Kenya [51] .", "The first description of RVA in chiroptera was recorded in feces of Eidolon helvum caught in Vihiga, Kenya [51] . Afterwards, several strains of RVA were detected by different molecular techniques involving chiroptera, in several countries, including Kenya (E. helvum), China (Rhinolophus hipposideros and Aselliscus stoliczkanus), France (Myotis mystacinus), Cameroon (E. helvum) and Brazil [31, [51] [52] [53] [54] [55] . The present study shows the occurrence of RVA in 17.98% of the chiroptera, being among the species Carollia perspicillata (37.5%), Desmodus rotundus (28.12%), Uroderma bilobata (15.6%), Artibeus lituratus (9.37%), Artibeus Planirostus (3.12%), Diaemus iyoug and Glossophagine (3.12%).", "The present study shows the occurrence of RVA in 17.98% of the chiroptera, being among the species Carollia perspicillata (37.5%), Desmodus rotundus (28.12%), Uroderma bilobata (15.6%), Artibeus lituratus (9.37%), Artibeus Planirostus (3.12%), Diaemus iyoug and Glossophagine (3.12%). Barquez et al. [56] reported that Desmodus rotundus is one of the three hematophagous species of the Phyllostomidae family, found throughout South America, Central America and Mexico.", "[56] reported that Desmodus rotundus is one of the three hematophagous species of the Phyllostomidae family, found throughout South America, Central America and Mexico. Of the positive chiroptera for RVA in the present study, a prevalence of 28.12% was of Desmodus rotundus. This species feeds on birds, can feed on mammals, mostly medium or large, facilitating the dissemination of viral spores among the community within the habitat, as observed in the present study.", "This species feeds on birds, can feed on mammals, mostly medium or large, facilitating the dissemination of viral spores among the community within the habitat, as observed in the present study. These findings show the importance of epidemiological data on the studied species due to the lack of studies involving species of neotropical chiroptera, and it is not possible to establish comparative parameters for these animals. Regarding the circulation of RVA in canines and birds, the prevalence was 53% and 29%, respectively.", "Regarding the circulation of RVA in canines and birds, the prevalence was 53% and 29%, respectively. Although in the Amazon region there are records of RVA, RVD, RVF and RVG that infect birds [57] [58] and RVD in migratory birds [59] , all were detected by RT-PCR assays differently from the present study which detected the RVA by RT-qPCR involving a variety of animal species. On the other hand, the prevalence in felines (16%) and pigs (22%) was lower, probably because there are few animals of these species in the region, as well as few creations.", "On the other hand, the prevalence in felines (16%) and pigs (22%) was lower, probably because there are few animals of these species in the region, as well as few creations. The study detected the presence of RVA in different species of animals both in areas near the home and in areas located in fragments of forest, characterized as forest remnants, since they were located in cities that suffered high environmental impacts due to vegetal extractivism, pasture formation for cattle breeding, exploitation of natural resources, and direct reflexes on the habitats of wild animals that can serve as virus sources, thus facilitating the dispersion of RVA among communities of coexisting animals. It is worth emphasizing that these animals have a greater contact with the human populations of the studied areas since they cohabit with the humans in the region, besides having a high flow of movement between the forest extracts and environments chosen for the present study.", "It is worth emphasizing that these animals have a greater contact with the human populations of the studied areas since they cohabit with the humans in the region, besides having a high flow of movement between the forest extracts and environments chosen for the present study. However, it is noteworthy that only in the communities of Santa Bárbara and Viseu were collected fecal specimens from asymptomatic humans for diarrhea and tested for RVA, but all were negative. It is notorious yet, the existence of different levels of degradation in the studied environments, considering the presented data.", "It is notorious yet, the existence of different levels of degradation in the studied environments, considering the presented data. The fragmentation of the forest generates many consequences on the Amazonian biota, being able to alter the diversity and the composition of the animal communities in the fragments and even to interfere in the ecological processes, without considering that the fragments of forest in the Amazon are influenced by the climate, possibly facilitating the dispersion of pathogens by the environment, since the wild animals detected in the present study are asymptomatic and have low viral load for RVA. The occurrence of RVA in this population of animals may explain the possibility of dispersion of viral strains, since there is a proximity to the human population, besides the biological characteristics of these species that may represent important sources for gastroenteric viruses, along with the fact that all animals were asymptomatic for diarrhea.", "The occurrence of RVA in this population of animals may explain the possibility of dispersion of viral strains, since there is a proximity to the human population, besides the biological characteristics of these species that may represent important sources for gastroenteric viruses, along with the fact that all animals were asymptomatic for diarrhea. Wild birds have unlimited flight capacity, were captured in an interface region between the peridomicile and forest fragments and it is believed that this region has not been influenced by anthropic activities such as those observed in the area of the present study. On the other hand, the breeding method for poultry and canines close to homes and the forest ecosystem, as they are created in the communities surveyed, probably facilitates direct contact with possible sources of contamination, since in the areas the use of septic tanks is deficient and sometimes non-existent, which may facilitate or even increase the risk of viral dispersion throughout the environment.", "On the other hand, the breeding method for poultry and canines close to homes and the forest ecosystem, as they are created in the communities surveyed, probably facilitates direct contact with possible sources of contamination, since in the areas the use of septic tanks is deficient and sometimes non-existent, which may facilitate or even increase the risk of viral dispersion throughout the environment. The high rates of increase and the analysis of land use in the researched areas may be important indicators of how these animals interact, since with deforestation, the populations of wild animals seek refuge in nearby communities facilitating the dispersal of infectious agents and the possible occurrence of carrier animals by direct contact or contamination of the local environment. To our knowledge, this is the first study in which a real-time PCR assay was applied for the detection of RVA involving a wide variety of domestic and wild animals, facilitating practical utility in epidemiological and molecular studies and assisting in a perspective in the elaboration of sanitary control and monitoring, preventing possible outbreaks in the studied communities.", "To our knowledge, this is the first study in which a real-time PCR assay was applied for the detection of RVA involving a wide variety of domestic and wild animals, facilitating practical utility in epidemiological and molecular studies and assisting in a perspective in the elaboration of sanitary control and monitoring, preventing possible outbreaks in the studied communities. The detection of positive animals was useful to monitor the infection of the agent in the animal population and to provide an early warning signal to predict an impending epidemic and a favorable risk for the human population, given the evidence of RVA circulation in the different forest fragments. In addition, the RT-qPCR assay may be a useful alternative for the differential diagnosis of RV in possible coexisting mixed infections clinically indistinguishable such as those caused by other viral strains that cause gastroenteritis such as: astrovirus, coronavirus, picobirnavirus, calicivirus, among others as observed in the studies of Jing et al.", "In addition, the RT-qPCR assay may be a useful alternative for the differential diagnosis of RV in possible coexisting mixed infections clinically indistinguishable such as those caused by other viral strains that cause gastroenteritis such as: astrovirus, coronavirus, picobirnavirus, calicivirus, among others as observed in the studies of Jing et al. [60] and Waruhiu et al. [61] .", "[60] and Waruhiu et al. [61] . Diarrhea associated with RVA infections in pigs is an important cause of increased mortality and economic losses in Europe.", "Diarrhea associated with RVA infections in pigs is an important cause of increased mortality and economic losses in Europe. The most prevalent genotypes isolated from feces of Belgian diarrheal and non-diarrheal piglets in 2012 [62] demonstrate a wide range of combinations of genotypes G / P including; G3P [6] , G4P [6] , G5P [6] , G4P [7] , G5P [7] , G9P [7] , G9P [13] and G9P [23] . On the other hand, in the present study it was possible to detect only P [6] genotype, since majority of samples was asymptomatic for diarrhea.", "On the other hand, in the present study it was possible to detect only P [6] genotype, since majority of samples was asymptomatic for diarrhea. Finding shows that different P genotypes of RVA strains interact with distinct blood group histological antigens (HBGA, ABOH, Lewis) and sialic acids via VP4 providing insight into the regional prevalence and increased zoonotic potential of some RVA of origin swine [63] . The genotype P [6] was identified in piglets in Brazil [64] and in Italy and Japan resembling genotype P [6] human [65, 66] .", "The genotype P [6] was identified in piglets in Brazil [64] and in Italy and Japan resembling genotype P [6] human [65, 66] . In the population of animals studied the zoonotic transmission can be frequent, since the animals live in contact with humans and in precarious sanitary conditions. In Brazil, this genotype was described in animal and human populations in studies of Luchs et al.", "In Brazil, this genotype was described in animal and human populations in studies of Luchs et al. [32] ; Honma et al. [67] ; Araújo et al. [68] ; Mascarenhas et al. [69] and Lorenzetti et al.", "[68] ; Mascarenhas et al. [69] and Lorenzetti et al. [70] such studies corroborate the importance of continuing to monitor genotypes to verify if uncommon strains or new strains are emerging and can infect animal populations or inter-species transmissions.", "[70] such studies corroborate the importance of continuing to monitor genotypes to verify if uncommon strains or new strains are emerging and can infect animal populations or inter-species transmissions. Regarding the genotype P [4] , itwas most detected in our samples in bats, dogs, swine and feline. This genotype is not common in animals, being more detected in human and environmental samples in various parts of the world and included our region [71] .", "This genotype is not common in animals, being more detected in human and environmental samples in various parts of the world and included our region [71] . It is important to emphasize that the indicators of environmental contamination in Brazil are significant and contribute to the possibility of human-animal transmission [71] . Such data need further investigation in later work to better characterize the interspecies transmission, since the occurrence of enteric viruses in different matrices demonstrates the anthropogenic impact of the exposed population around and points to the potential risk of infection by the possible exposure of individuals susceptible.", "Such data need further investigation in later work to better characterize the interspecies transmission, since the occurrence of enteric viruses in different matrices demonstrates the anthropogenic impact of the exposed population around and points to the potential risk of infection by the possible exposure of individuals susceptible. Our findings may be useful for tracking fecal contamination in the environment using animals as possible sources thus minimizing the risk of infection by exposure to susceptible individuals, in this case different animal species or even human populations. RVA were detected in wild and domestic animals using a RT-qPCR assay that analyzed samples that had low viral load for RVA.", "RVA were detected in wild and domestic animals using a RT-qPCR assay that analyzed samples that had low viral load for RVA. Although the samples are asymptomatic for diarrhea, it is necessary to conduct strategies for the monitoring and control of the animals in the areas studied in the human population as well as in other species of animals, as well as the implementation of preventive measures aimed at future outbreaks in communities animals in the resident population in these impacted areas. Therefore, the present study is unprecedented in the region and in the country in relation to the research of RVA in wild animals.", "Therefore, the present study is unprecedented in the region and in the country in relation to the research of RVA in wild animals. It is noteworthy that, although the quality of the analyzed samples is characterized as low detectable viral load, the technique presented a good analytical response in the detection of the source animals for RVA, facilitating the selection of the samples for future genetic characterization tests." ]

[ "Acute gastroenteritis is one of the main causes of mortality in humans and young animals. Domestic and mainly wild animals such as bats, small rodents and birds are highly diversified animals in relation to their habitats and ecological niches and are widely distributed geographically in environments of forest fragmentation in some areas of the Amazon, being considered important sources for viruses that affect humans and other animals. Due to the anthropical activities, these animals changed their natural habitat and adapted to urbanized environments, thus representing risks to human and animal health.", "Due to the anthropical activities, these animals changed their natural habitat and adapted to urbanized environments, thus representing risks to human and animal health. Although the knowledge of the global diversity of enteric viruses is scarce, there are reports demonstrating the detection of rotavirus in domestic animals and animals of productive systems, such as bovines and pigs. The present study investigated the prevalence of Rotavirus A in 648 fecal samples of different animal species from the northeastern mesoregion of the state of Pará, Brazil, which is characterized as an urbanized area with forest fragments.", "The present study investigated the prevalence of Rotavirus A in 648 fecal samples of different animal species from the northeastern mesoregion of the state of Pará, Brazil, which is characterized as an urbanized area with forest fragments. The fecal specimens were collected from October 2014 to April 2016 and subjected to a Qualitative Real-Time Polymerase Chain Reaction (RT-qPCR), using the NSP3 gene as a target. It was observed that 27.5% (178/648) of the samples presented positive results for RVA, with 178 samples distributed in birds (23.6%), canines (21.35%), chiropterans (17.98%), bovines (14.6%), horses (8.43%), small rodents (6.74%), pigs (3.93%) and felines (3.37%), demonstrating the circulation of RVA in domestic animals and suggesting that such proximity could cause transmissions between different species and the occurrence of rearrangements in the genome of RVA as already described in the literature, associated to the traces of environmental degradation in the studied areas.", "It was observed that 27.5% (178/648) of the samples presented positive results for RVA, with 178 samples distributed in birds (23.6%), canines (21.35%), chiropterans (17.98%), bovines (14.6%), horses (8.43%), small rodents (6.74%), pigs (3.93%) and felines (3.37%), demonstrating the circulation of RVA in domestic animals and suggesting that such proximity could cause transmissions between different species and the occurrence of rearrangements in the genome of RVA as already described in the literature, associated to the traces of environmental degradation in the studied areas. Text: Emerging and reemerging infectious diseases are increasing each year in several countries, with an impact both on human populations and on domestic and wild animals living in areas with considerable forest remnants [1] . Most of these diseases are of viral origin, suggesting the emergence and reemergence of viruses that are triggered by human activities that modify the environment [2] .", "Most of these diseases are of viral origin, suggesting the emergence and reemergence of viruses that are triggered by human activities that modify the environment [2] . The populations of wild animals that inhabit forest fragments are strategic groups for studies of public health and the transmission of zoonosis, given that they act as indicators in the assistance and intervention in the human populations, aiming at the prevention of outbreaks and epidemics [3] . Acute gastroenteritis can be caused by infection in the gastrointestinal tract, caused by different infectious or parasite agents [4] [5] [6] [7] .", "Acute gastroenteritis can be caused by infection in the gastrointestinal tract, caused by different infectious or parasite agents [4] [5] [6] [7] . They represent one of the main causes of mortality in humans, and in young animals, counting for about 25% of mortality [8] . Rotavirus is widely distributed in animals, which act as sources of rotavirus emergent strains, with these animals acting in the transmission between species and through reassortment leading to the emergence of new strains which have been reported in human infections [9] [10] [11] [12] .", "Rotavirus is widely distributed in animals, which act as sources of rotavirus emergent strains, with these animals acting in the transmission between species and through reassortment leading to the emergence of new strains which have been reported in human infections [9] [10] [11] [12] . The rotavirus (RV) belongs to the Reoviridae family and comprises nine species known as Rotavirus group A to I, with a recent proposal of the J species [13, 14] . Rotavirus A (RVA) is widespread worldwide and predominantly infects humans, bovines and other mammal species, as well as birds [15] .", "Rotavirus A (RVA) is widespread worldwide and predominantly infects humans, bovines and other mammal species, as well as birds [15] . They have a double-stranded ribonucleic acid (dsRNA) genome, divided into 11 segments coding for structural proteins (VP1-VP4, VP6 and VP7) and nonstructural (NSP1-NSP5/NSP6) proteins [16, 17] . There are records of a close relationship between Amazonian wildlife and human populations [18] , and this interaction is the effect of anthropogenic urbanization activities that result in the deforestation of forest areas, causing the degradation of previously isolated sites such as caves and small caves, a continuous and nature progressive process that has led not only to changes in wildlife habitats but also to a greater relationship with human populations in rural and urban environments, contributing to the occurrence and emergence of diseases different from what normally occurs in endemic regions [19] [20] [21] [22] .", "There are records of a close relationship between Amazonian wildlife and human populations [18] , and this interaction is the effect of anthropogenic urbanization activities that result in the deforestation of forest areas, causing the degradation of previously isolated sites such as caves and small caves, a continuous and nature progressive process that has led not only to changes in wildlife habitats but also to a greater relationship with human populations in rural and urban environments, contributing to the occurrence and emergence of diseases different from what normally occurs in endemic regions [19] [20] [21] [22] . Although the results of RVA have already been described globally [12, [23] [24] [25] [26] [27] [28] [29] [30] , in Brazil, the occurrence, diversity and role of rotavirus in these animals are still poorly studied, considering the large number of present species [4, [31] [32] [33] [34] . In the Brazilian Amazon, especially in the state of Pará, the city of Belém and Northeast metropolitan mesoregions are some of the areas with the highest indexes of environmental changes [35] , which are concentrated, along with the fact that the knowledge of the global diversity of enteric virus in animals is scarce [36] .", "In the Brazilian Amazon, especially in the state of Pará, the city of Belém and Northeast metropolitan mesoregions are some of the areas with the highest indexes of environmental changes [35] , which are concentrated, along with the fact that the knowledge of the global diversity of enteric virus in animals is scarce [36] . Therefore, it is important to monitor the health of domestic and wild animals in their natural habitat, especially in areas with anthropic alterations that have an interface with rural communities and enterprises, in order to investigate the occurrence of RVA in this population. These communities are ecologically complex, because they have multiple hosts and endless pathogens that may eventually circulate in contiguous urban centers, in addition to the fact that it should also be considered that there is still a lack of studies showing the significance of these viruses infecting this population, as in the context of epidemiological surveillance, these animals become important, since they can be considered as natural sources, with the possibility of transmission to humans [37] [38] [39] .", "These communities are ecologically complex, because they have multiple hosts and endless pathogens that may eventually circulate in contiguous urban centers, in addition to the fact that it should also be considered that there is still a lack of studies showing the significance of these viruses infecting this population, as in the context of epidemiological surveillance, these animals become important, since they can be considered as natural sources, with the possibility of transmission to humans [37] [38] [39] . The qualitative real-time polymerase chain reaction (qRT-PCR) used the NSP3 gene and the TaqMan probe from a highly conserved region of the rotavirus non-structural protein 3 (NSP3), which was previously used in samples from human origin and with low viral loads \n\nPrecipitation data were obtained from The Brazilian National Institute of Meteorology (Inmet for the years of capture in the Expedito Ribeiro Settlement (2014) and Açailândia (2015) of the Data Collection Platforms (PCDs) of Belém, located 50 km from Santa Bárbara do Pará, and Tracuateua, located 50 km from Peixe-Boi and 100 km from Viseu. Garmin GPSMap 64s Global Positioning System (GPS) coordinates were collected in the field.", "Garmin GPSMap 64s Global Positioning System (GPS) coordinates were collected in the field. The municipal boundaries were obtained on the website of the Brazilian Institute of Geography and Statistics (IBGE) ( and data on deforestation and land use were obtained from the PRODES [43] and TerraClass [44] Projects. PRODES has annual data in digital format since 2000 and TerraClass presents biannual data since 2004.", "PRODES has annual data in digital format since 2000 and TerraClass presents biannual data since 2004. The satellite image was generated using the sensor Sentinel 2 of the European Space Agency (ESA) ( web/sentinel/user-guides/sentinel-2-msi) with Open Access CC-BY License ( from the years of 2017 and 2018. All the data obtained was stored in a Geographic Database (BDG).", "All the data obtained was stored in a Geographic Database (BDG). The BDG was imported/ stored in a GIS for the editing of the graphic elements, establishment of topological relations between the elements and their respective attributes, spatial analysis and visualization of the result through thematic maps. For the present study, forest fragments of similar size, shape and Phyto physiology were chosen, considering an open peri urban matrix with similar soil use.", "For the present study, forest fragments of similar size, shape and Phyto physiology were chosen, considering an open peri urban matrix with similar soil use. The selected fragments were distributed within the mesoregions studied, and in each selected fragment fecal samples were randomly collected from domestic and wild animals [45] . Soil use classes were obtained from the TerraClass data mosaic from 2004 to 2016, because the study sites were in an area with a high cloud presence, which prevented observation (the area was not observed).", "Soil use classes were obtained from the TerraClass data mosaic from 2004 to 2016, because the study sites were in an area with a high cloud presence, which prevented observation (the area was not observed). The data processing, interpretation, visualization and spatial analysis were performed in ArcGIS software ( For the analysis of data related to the determination of the richness, composition and abundance of the fauna of the animals studied in the study area, considering the collection methods adopted and the species available in each city, each sample was considered as an independent sample. The richness of wild fauna and domestic animals was determined by the total number of species including all collection methods, and the similarity of species was made by the chi-square analysis between the samples of the different treatments with the aid of the EstimateS 8.0 software [46] .", "The richness of wild fauna and domestic animals was determined by the total number of species including all collection methods, and the similarity of species was made by the chi-square analysis between the samples of the different treatments with the aid of the EstimateS 8.0 software [46] . For the calculation of the Test T, the Statistica software was used, and the indices of infected animals in the two environments (forest fragment and peridomicile) were calculated for each treatment sampled by collection area, using the software Past 1.92. Aiming at comparing the values of the diversity indexes through the paired test, as well as the descriptive analysis of the anthropic effects [47] .", "Aiming at comparing the values of the diversity indexes through the paired test, as well as the descriptive analysis of the anthropic effects [47] . The data obtained for the occurrence of RVA and the questionnaires was inserted into a database for a descriptive analysis of the epidemiological profile of the animal population in the three forest ecosystems studied. In this analysis, descriptive statistical treatments were carried out, using customized \"row-columns\" type charts, referring to the data, in order to characterize the sample and quantify the results using absolute frequency values using the chi-square test and the Test T.\n\nPopulation study, collection of clinical specimens and laboratory methodology.", "In this analysis, descriptive statistical treatments were carried out, using customized \"row-columns\" type charts, referring to the data, in order to characterize the sample and quantify the results using absolute frequency values using the chi-square test and the Test T.\n\nPopulation study, collection of clinical specimens and laboratory methodology. The flying animals (wild birds and chiroptera) were captured using mist nets which were opened at dawn (4:00 a.m.) and closed in the morning (9:00 a.m.) and were inspected every one hour until the closing, with a sampling effort of 15 days. This research was approved by National All procedures with animals were performed by veterinarians, being birds and bats identified and released at the same capture site.", "This research was approved by National All procedures with animals were performed by veterinarians, being birds and bats identified and released at the same capture site. The fecal specimens were collected by stimulation of the rectal ampulla with the use of a \"Zaragatoa\", packed in cryogenic vials, identified, stored in liquid nitrogen, and later sent to the Laboratory. Wild animals (small non-flying mammals) were trapped within live-containment traps of the Tomahawk cage (size 45x16x16cm) and Sherman type aluminum (size 30x9x8cm).", "Wild animals (small non-flying mammals) were trapped within live-containment traps of the Tomahawk cage (size 45x16x16cm) and Sherman type aluminum (size 30x9x8cm). In each sample plot, 61 traps were distributed, 20 Shermans and 41 Tomahawks being baited with a mixture made with peanut paste, sardines, cod liver oil and corn meal, as well as fruit like banana, apple and pineapple. All the traps used were inspected daily in the morning, the baits being exchanged when necessary and later after the capture in bags of cloth and at least five specimens of each species were chosen for the collection of biological material.", "All the traps used were inspected daily in the morning, the baits being exchanged when necessary and later after the capture in bags of cloth and at least five specimens of each species were chosen for the collection of biological material. The wild animals were sedated with a combination of ketamine 20mg/kg and xylazine 2mg/kg intramuscularly and subsequently, euthanized with anesthetic overdose of 2% lidocaine in the foramen magnum, according to the recommendation of the National Council for the Control of Animal Experimentation (CONCEA). From October 2014 to April 2016, 1,282 fecal samples were collected from wild and domestic animals.", "From October 2014 to April 2016, 1,282 fecal samples were collected from wild and domestic animals. Amongthese, 648 (50.5%) samples were randomly selected for RVA research and handled in Level Three Biosafety Laboratory (NB3). The viral genome was extracted using the TRIZOL LS REAGENT protocol (INVITRO-GEN, USA/KIT QIAGEN), following the manufacturer's recommendations, with minor adaptation according to the protocol described in the supplemental data.", "The viral genome was extracted using the TRIZOL LS REAGENT protocol (INVITRO-GEN, USA/KIT QIAGEN), following the manufacturer's recommendations, with minor adaptation according to the protocol described in the supplemental data. The qRT-PCR was conducted according to Zeng et al. [40] for the detection of RVA using the NSP3 segment of RVA as the target gene sequence.", "[40] for the detection of RVA using the NSP3 segment of RVA as the target gene sequence. The assay was conducted in a mixture containing: RNAse-free H 2 O, TaqMan RT-PCR Mix (2x), TAqMan RT Enzyme Mix (40x), primers for the NSP3 gene, Primer NSP3 Forward (20mM), Primer NSP3 Reverse (20mM), probe NSP3 S (10nm), Template (RNA) 3μL, having a total reaction volume of 17μL and reverse transcription cycling of 50˚C, 30 minutes, denaturation of 95˚C, 10 minutes, annealing of 45 cycles of 95˚C, 15 seconds and extension of 60˚C, 1 minute. The analyzes were considered positive when presenting the cycle threshold (CT) � 40.", "The analyzes were considered positive when presenting the cycle threshold (CT) � 40. In order to guarantee a reliable test result, the measurements of contamination control were performed with the use of positive animal control (SA11 prototype) and a negative control (ultrapure water). All RVA-positive samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) according to Mijatovic et al [41] to genotyping low viral loads samples.", "All RVA-positive samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) according to Mijatovic et al [41] to genotyping low viral loads samples. First round was performed with consensus primers N-VP4F1/N-VP4R1 and the Nested-PCR was conducted with N-VP4F2/N-VP4R2 primers to amplify VP4 gene. Amplicons were purified and sequencing for VP4 gene using the same primers of Nested-PCR.", "Amplicons were purified and sequencing for VP4 gene using the same primers of Nested-PCR. The sequences were collected from an automated ABI Prism 3130xl DNA sequencer (Applied Biosystems). The sequence fragments were assembled and edited using the Geneious Bioinformatics software platform v.8.1.7.", "The sequence fragments were assembled and edited using the Geneious Bioinformatics software platform v.8.1.7. Posteriorly, the data were compared with othersequences from the National Center for Biotechnology Information GenBank database using BLAST alignment tool to elucidate the RVA genotype of the samples. From October 2014 to April 2016, a total of 648 fecal samples of wild and domestic animals belonging to three forest fragments areas were tested for the NSP3 gene by qualitative qPCR, and 178 (27.5%) were positive for RVA, distributed among the species: birds (23.6%), canines (21.35%), bats (17.98%), cattle (14.6%), horses (8.43%), small rodents (6.74%), swine (3.93%) and felines (3.37%).", "From October 2014 to April 2016, a total of 648 fecal samples of wild and domestic animals belonging to three forest fragments areas were tested for the NSP3 gene by qualitative qPCR, and 178 (27.5%) were positive for RVA, distributed among the species: birds (23.6%), canines (21.35%), bats (17.98%), cattle (14.6%), horses (8.43%), small rodents (6.74%), swine (3.93%) and felines (3.37%). The CT interval ranged from 28. 47 It was possible to detect viral strains in all genders of animals studied and in the harvesting period none of the animals showed signs of acute infection and / or diarrhea.", "47 It was possible to detect viral strains in all genders of animals studied and in the harvesting period none of the animals showed signs of acute infection and / or diarrhea. Rotavirus A (RVA) detected in the present study of wild and domestic animals belonging to the three areas of forest fragment, according to Fig 2. In relation to the evaluated bovines, only in the city of Viseu, these species were studied because they were created extensively.", "In relation to the evaluated bovines, only in the city of Viseu, these species were studied because they were created extensively. In addition, most of the animals were young with ages varying from 1 day to 8 yearsold, history of deficient vaccination, lack of technical assistance and raised in the form of subsistence. The animals showed no symptoms of diarrhea, only low weight performance and poor sanitary management status.", "The animals showed no symptoms of diarrhea, only low weight performance and poor sanitary management status. In relation to chiroptera, 32 (17.98%) positive samples for RVA were distributed among Carollia perspicillata species, with 12 (37.5%) being all adults, 9 (28.12%) Desmodus rotundus samples (4 young and 5 adults), 5 (15.6%) of Uroderma bilobata (15.62%), 3 (9.37%) of Artibeus lituratus and the species Artibeus Planirostus, Diaemus iyoug and Glossophagine with 1 (3.12%) each. These animals came from areas of forest fragments located near bovine and equine farms, in addition to inhabiting small chicken farms.", "These animals came from areas of forest fragments located near bovine and equine farms, in addition to inhabiting small chicken farms. Fig 3 shows the results obtained for all the species of animals investigated in the forest fragment as well as in the peridomicillus area. The anthropic variables were analyzed for the three cities studied, as well as the use of the soil within the range of the animals, obeying the domicile, the peridomicile and the forest fragment where the traps of small rodents, birds and various species of animals were captured (Fig 4 and Fig 5) .", "The anthropic variables were analyzed for the three cities studied, as well as the use of the soil within the range of the animals, obeying the domicile, the peridomicile and the forest fragment where the traps of small rodents, birds and various species of animals were captured (Fig 4 and Fig 5) . Considering the factors related to the anthropic activities in the three studied areas within the three cities of the present study, it was observed that the city of Santa Bárbara is the one that has a better area of preserved forest and the city of Viseu a smaller area. However, in the city of Santa Bárbara, a greater concentration of occupations was observed around the area of forest fragment.", "However, in the city of Santa Bárbara, a greater concentration of occupations was observed around the area of forest fragment. It was observed in this chosen area of the city, the presence of different families living in a rural settlement, surviving from the exploitation of forest resources and the creation of small animals for subsistence, such as poultry and fish farming, as well as family farming products. The breeding of animals in native pastures was only observed in the cities of Peixe Boi and Viseu.", "The breeding of animals in native pastures was only observed in the cities of Peixe Boi and Viseu. Extensive livestock farming was practiced with beef cattle, equines for work and small animals (swine and goats). In relation to the most preserved pasture area, the city of Peixe Boi had the largest area, according to the data shown in Fig 5, however, in the city of Viseu, a higher regeneration was observed in the pastures during the period of the study, with significant secondary vegetation.", "In relation to the most preserved pasture area, the city of Peixe Boi had the largest area, according to the data shown in Fig 5, however, in the city of Viseu, a higher regeneration was observed in the pastures during the period of the study, with significant secondary vegetation. When comparing the climates of the three areas it was observed that the predominant climate is megathermal and humid with average annual temperature around 27˚C. The months of October, November and December are the hottest, with temperatures between 32˚C and 34˚C and absolute maximums around 41˚C.", "The months of October, November and December are the hottest, with temperatures between 32˚C and 34˚C and absolute maximums around 41˚C. Annual rainfall is quite high, generally around 2,350 mm, but strongly concentrated from January to June (80%). From September to December, on the contrary, rainfall is rare, about 7%, with a short dry season, of moderate water deficit in those months.", "From September to December, on the contrary, rainfall is rare, about 7%, with a short dry season, of moderate water deficit in those months. The relative humidity of the average air oscillates around 85%, as shown in Fig 6 [48] . The description of the accumulated precipitation in the year of capture of the fecal specimens compared to the Climatological Normals (CLINO) for the period from 1961-1990 of the PCDs closest to the locations of the Expedito Ribeiro / Santa Bárbara settlement (Belém PCD), Vila Ananim / Peixe-Boi and Açaiteua / Viseu (Tracauateua PCD) show the frequency of rainfall in the regions, which facilitates the renewal of the pastures and the regeneration of the impacted forests, being an important indicator of the reduction of the damages caused by deforestation in the region.", "The description of the accumulated precipitation in the year of capture of the fecal specimens compared to the Climatological Normals (CLINO) for the period from 1961-1990 of the PCDs closest to the locations of the Expedito Ribeiro / Santa Bárbara settlement (Belém PCD), Vila Ananim / Peixe-Boi and Açaiteua / Viseu (Tracauateua PCD) show the frequency of rainfall in the regions, which facilitates the renewal of the pastures and the regeneration of the impacted forests, being an important indicator of the reduction of the damages caused by deforestation in the region. The average deforestation index in the three study areas was calculated from data obtained from INPE information systems. It was observed that in the years of 2013 to 2014 there were no changes in these regions; in the period from 2014 to 2015 about 4.1% of the city of Viseu was changed and 1.6% of the city of Peixe Boi.", "It was observed that in the years of 2013 to 2014 there were no changes in these regions; in the period from 2014 to 2015 about 4.1% of the city of Viseu was changed and 1.6% of the city of Peixe Boi. In relation to the period of 2016, great changes were observed in Peixe Boi (79%) and in the city of Viseu (70%), thus demonstrating that changes in the natural ecosystem may be associated with the frequencies for RVA in the studied areas, according to Fig 7. When assessing the infected animals in relation to the uninfected animals in both the forest fragment and the peridomicile, considering as animals of the forest fragment the birds, the chiroptera and the small rodents and as animals of the peridomicile the canines, bovines, pigs, felines and horses, a percentage of 37.07% infected peri domestic animals (86/232) and 22.12% infected forest fragment animals (92/416) were obtained.", "When assessing the infected animals in relation to the uninfected animals in both the forest fragment and the peridomicile, considering as animals of the forest fragment the birds, the chiroptera and the small rodents and as animals of the peridomicile the canines, bovines, pigs, felines and horses, a percentage of 37.07% infected peri domestic animals (86/232) and 22.12% infected forest fragment animals (92/416) were obtained. Applying the selected statistical analysis, a Pearson x2 Chi-square value was obtained: 16.7159, df = 1 and p <0.001, meaning that the hypothesis was corroborated, that is, the greater the degradation of the environment, the more likely it will be the search for food by wild animals in adjacent areas, or in the edge of the forest or even in the peri domiciliary region. In this sense, the possibility of contagion with other species of animals, even humans, should be considered because of the capacity of the rotavirus to be transmitted via the fecal / oral route or through direct contact with the environment.", "In this sense, the possibility of contagion with other species of animals, even humans, should be considered because of the capacity of the rotavirus to be transmitted via the fecal / oral route or through direct contact with the environment. It is important to point out that the animals detected in this study are important sources of viral strains. A total of 80 stool samples were selected, reextracted and analyzed using PCR for the VP4 gene.", "A total of 80 stool samples were selected, reextracted and analyzed using PCR for the VP4 gene. Eight strains (10%) were positive for VP4 gene, being 2 strains bellowed to P [6] genotype and 6 to P[4]-type, according to \n\nIn the present study, RVA was detected circulating in 27.5% of the animals; 36% in domestic animals and 64% in wild animals, providing a unique dataset with qRT-PCR detecting a low viral load of RVA in different species, which further correlates with the deforestation index. These data are important because there is a lack of tests for RVA diagnosis in animals, since the current methods of RVA detection does not always detect in these populations [8] .", "These data are important because there is a lack of tests for RVA diagnosis in animals, since the current methods of RVA detection does not always detect in these populations [8] . With the advent of real-time PCR (qPCR), there was an exponential growth, compared to conventional PCR essays, since its superior accuracy, sensitivity and specificity is remarkable, and it is Rotavirus A in wild and domestic animals possible to detect RVA in a variety of animal species using NSP3 gene [49] . The sensitivity of RT-qPCR significantly improved the rate of RVA detection in clinical samples from animals and in this context, the present study proposed an interesting study metrics using virus spreading in the wild animals which inhabit forest fragments to indicate human population interventions, with the goal of preventing the virus outbreaks leveraged on the unique geographic characteristics of Brazil and its large number of species in Amazon.", "The sensitivity of RT-qPCR significantly improved the rate of RVA detection in clinical samples from animals and in this context, the present study proposed an interesting study metrics using virus spreading in the wild animals which inhabit forest fragments to indicate human population interventions, with the goal of preventing the virus outbreaks leveraged on the unique geographic characteristics of Brazil and its large number of species in Amazon. Currently, no data have been described in the literature regarding the RVA detection using real-time qPCR technique in a wide variety of wild animal species. However, a study by Soltan et al.", "However, a study by Soltan et al. [50] conducted with horses and cattle detected RVA by RT-PCR, commercial RT-PCR and RT-qPCR in 36.7%, 51.4% and 56.9% respectively, differently from the present study that showed higher positivity for chiropterans (17.98%), canines (21.35%), birds (23.6%) and cattle (14.6%). The first description of RVA in chiroptera was recorded in feces of Eidolon helvum caught in Vihiga, Kenya [51] .", "The first description of RVA in chiroptera was recorded in feces of Eidolon helvum caught in Vihiga, Kenya [51] . Afterwards, several strains of RVA were detected by different molecular techniques involving chiroptera, in several countries, including Kenya (E. helvum), China (Rhinolophus hipposideros and Aselliscus stoliczkanus), France (Myotis mystacinus), Cameroon (E. helvum) and Brazil [31, [51] [52] [53] [54] [55] . The present study shows the occurrence of RVA in 17.98% of the chiroptera, being among the species Carollia perspicillata (37.5%), Desmodus rotundus (28.12%), Uroderma bilobata (15.6%), Artibeus lituratus (9.37%), Artibeus Planirostus (3.12%), Diaemus iyoug and Glossophagine (3.12%).", "The present study shows the occurrence of RVA in 17.98% of the chiroptera, being among the species Carollia perspicillata (37.5%), Desmodus rotundus (28.12%), Uroderma bilobata (15.6%), Artibeus lituratus (9.37%), Artibeus Planirostus (3.12%), Diaemus iyoug and Glossophagine (3.12%). Barquez et al. [56] reported that Desmodus rotundus is one of the three hematophagous species of the Phyllostomidae family, found throughout South America, Central America and Mexico.", "[56] reported that Desmodus rotundus is one of the three hematophagous species of the Phyllostomidae family, found throughout South America, Central America and Mexico. Of the positive chiroptera for RVA in the present study, a prevalence of 28.12% was of Desmodus rotundus. This species feeds on birds, can feed on mammals, mostly medium or large, facilitating the dissemination of viral spores among the community within the habitat, as observed in the present study.", "This species feeds on birds, can feed on mammals, mostly medium or large, facilitating the dissemination of viral spores among the community within the habitat, as observed in the present study. These findings show the importance of epidemiological data on the studied species due to the lack of studies involving species of neotropical chiroptera, and it is not possible to establish comparative parameters for these animals. Regarding the circulation of RVA in canines and birds, the prevalence was 53% and 29%, respectively.", "Regarding the circulation of RVA in canines and birds, the prevalence was 53% and 29%, respectively. Although in the Amazon region there are records of RVA, RVD, RVF and RVG that infect birds [57] [58] and RVD in migratory birds [59] , all were detected by RT-PCR assays differently from the present study which detected the RVA by RT-qPCR involving a variety of animal species. On the other hand, the prevalence in felines (16%) and pigs (22%) was lower, probably because there are few animals of these species in the region, as well as few creations.", "On the other hand, the prevalence in felines (16%) and pigs (22%) was lower, probably because there are few animals of these species in the region, as well as few creations. The study detected the presence of RVA in different species of animals both in areas near the home and in areas located in fragments of forest, characterized as forest remnants, since they were located in cities that suffered high environmental impacts due to vegetal extractivism, pasture formation for cattle breeding, exploitation of natural resources, and direct reflexes on the habitats of wild animals that can serve as virus sources, thus facilitating the dispersion of RVA among communities of coexisting animals. It is worth emphasizing that these animals have a greater contact with the human populations of the studied areas since they cohabit with the humans in the region, besides having a high flow of movement between the forest extracts and environments chosen for the present study.", "It is worth emphasizing that these animals have a greater contact with the human populations of the studied areas since they cohabit with the humans in the region, besides having a high flow of movement between the forest extracts and environments chosen for the present study. However, it is noteworthy that only in the communities of Santa Bárbara and Viseu were collected fecal specimens from asymptomatic humans for diarrhea and tested for RVA, but all were negative. It is notorious yet, the existence of different levels of degradation in the studied environments, considering the presented data.", "It is notorious yet, the existence of different levels of degradation in the studied environments, considering the presented data. The fragmentation of the forest generates many consequences on the Amazonian biota, being able to alter the diversity and the composition of the animal communities in the fragments and even to interfere in the ecological processes, without considering that the fragments of forest in the Amazon are influenced by the climate, possibly facilitating the dispersion of pathogens by the environment, since the wild animals detected in the present study are asymptomatic and have low viral load for RVA. The occurrence of RVA in this population of animals may explain the possibility of dispersion of viral strains, since there is a proximity to the human population, besides the biological characteristics of these species that may represent important sources for gastroenteric viruses, along with the fact that all animals were asymptomatic for diarrhea.", "The occurrence of RVA in this population of animals may explain the possibility of dispersion of viral strains, since there is a proximity to the human population, besides the biological characteristics of these species that may represent important sources for gastroenteric viruses, along with the fact that all animals were asymptomatic for diarrhea. Wild birds have unlimited flight capacity, were captured in an interface region between the peridomicile and forest fragments and it is believed that this region has not been influenced by anthropic activities such as those observed in the area of the present study. On the other hand, the breeding method for poultry and canines close to homes and the forest ecosystem, as they are created in the communities surveyed, probably facilitates direct contact with possible sources of contamination, since in the areas the use of septic tanks is deficient and sometimes non-existent, which may facilitate or even increase the risk of viral dispersion throughout the environment.", "On the other hand, the breeding method for poultry and canines close to homes and the forest ecosystem, as they are created in the communities surveyed, probably facilitates direct contact with possible sources of contamination, since in the areas the use of septic tanks is deficient and sometimes non-existent, which may facilitate or even increase the risk of viral dispersion throughout the environment. The high rates of increase and the analysis of land use in the researched areas may be important indicators of how these animals interact, since with deforestation, the populations of wild animals seek refuge in nearby communities facilitating the dispersal of infectious agents and the possible occurrence of carrier animals by direct contact or contamination of the local environment. To our knowledge, this is the first study in which a real-time PCR assay was applied for the detection of RVA involving a wide variety of domestic and wild animals, facilitating practical utility in epidemiological and molecular studies and assisting in a perspective in the elaboration of sanitary control and monitoring, preventing possible outbreaks in the studied communities.", "To our knowledge, this is the first study in which a real-time PCR assay was applied for the detection of RVA involving a wide variety of domestic and wild animals, facilitating practical utility in epidemiological and molecular studies and assisting in a perspective in the elaboration of sanitary control and monitoring, preventing possible outbreaks in the studied communities. The detection of positive animals was useful to monitor the infection of the agent in the animal population and to provide an early warning signal to predict an impending epidemic and a favorable risk for the human population, given the evidence of RVA circulation in the different forest fragments. In addition, the RT-qPCR assay may be a useful alternative for the differential diagnosis of RV in possible coexisting mixed infections clinically indistinguishable such as those caused by other viral strains that cause gastroenteritis such as: astrovirus, coronavirus, picobirnavirus, calicivirus, among others as observed in the studies of Jing et al.", "In addition, the RT-qPCR assay may be a useful alternative for the differential diagnosis of RV in possible coexisting mixed infections clinically indistinguishable such as those caused by other viral strains that cause gastroenteritis such as: astrovirus, coronavirus, picobirnavirus, calicivirus, among others as observed in the studies of Jing et al. [60] and Waruhiu et al. [61] .", "[60] and Waruhiu et al. [61] . Diarrhea associated with RVA infections in pigs is an important cause of increased mortality and economic losses in Europe.", "Diarrhea associated with RVA infections in pigs is an important cause of increased mortality and economic losses in Europe. The most prevalent genotypes isolated from feces of Belgian diarrheal and non-diarrheal piglets in 2012 [62] demonstrate a wide range of combinations of genotypes G / P including; G3P [6] , G4P [6] , G5P [6] , G4P [7] , G5P [7] , G9P [7] , G9P [13] and G9P [23] . On the other hand, in the present study it was possible to detect only P [6] genotype, since majority of samples was asymptomatic for diarrhea.", "On the other hand, in the present study it was possible to detect only P [6] genotype, since majority of samples was asymptomatic for diarrhea. Finding shows that different P genotypes of RVA strains interact with distinct blood group histological antigens (HBGA, ABOH, Lewis) and sialic acids via VP4 providing insight into the regional prevalence and increased zoonotic potential of some RVA of origin swine [63] . The genotype P [6] was identified in piglets in Brazil [64] and in Italy and Japan resembling genotype P [6] human [65, 66] .", "The genotype P [6] was identified in piglets in Brazil [64] and in Italy and Japan resembling genotype P [6] human [65, 66] . In the population of animals studied the zoonotic transmission can be frequent, since the animals live in contact with humans and in precarious sanitary conditions. In Brazil, this genotype was described in animal and human populations in studies of Luchs et al.", "In Brazil, this genotype was described in animal and human populations in studies of Luchs et al. [32] ; Honma et al. [67] ; Araújo et al. [68] ; Mascarenhas et al. [69] and Lorenzetti et al.", "[68] ; Mascarenhas et al. [69] and Lorenzetti et al. [70] such studies corroborate the importance of continuing to monitor genotypes to verify if uncommon strains or new strains are emerging and can infect animal populations or inter-species transmissions.", "[70] such studies corroborate the importance of continuing to monitor genotypes to verify if uncommon strains or new strains are emerging and can infect animal populations or inter-species transmissions. Regarding the genotype P [4] , itwas most detected in our samples in bats, dogs, swine and feline. This genotype is not common in animals, being more detected in human and environmental samples in various parts of the world and included our region [71] .", "This genotype is not common in animals, being more detected in human and environmental samples in various parts of the world and included our region [71] . It is important to emphasize that the indicators of environmental contamination in Brazil are significant and contribute to the possibility of human-animal transmission [71] . Such data need further investigation in later work to better characterize the interspecies transmission, since the occurrence of enteric viruses in different matrices demonstrates the anthropogenic impact of the exposed population around and points to the potential risk of infection by the possible exposure of individuals susceptible.", "Such data need further investigation in later work to better characterize the interspecies transmission, since the occurrence of enteric viruses in different matrices demonstrates the anthropogenic impact of the exposed population around and points to the potential risk of infection by the possible exposure of individuals susceptible. Our findings may be useful for tracking fecal contamination in the environment using animals as possible sources thus minimizing the risk of infection by exposure to susceptible individuals, in this case different animal species or even human populations. RVA were detected in wild and domestic animals using a RT-qPCR assay that analyzed samples that had low viral load for RVA.", "RVA were detected in wild and domestic animals using a RT-qPCR assay that analyzed samples that had low viral load for RVA. Although the samples are asymptomatic for diarrhea, it is necessary to conduct strategies for the monitoring and control of the animals in the areas studied in the human population as well as in other species of animals, as well as the implementation of preventive measures aimed at future outbreaks in communities animals in the resident population in these impacted areas. Therefore, the present study is unprecedented in the region and in the country in relation to the research of RVA in wild animals.", "Therefore, the present study is unprecedented in the region and in the country in relation to the research of RVA in wild animals. It is noteworthy that, although the quality of the analyzed samples is characterized as low detectable viral load, the technique presented a good analytical response in the detection of the source animals for RVA, facilitating the selection of the samples for future genetic characterization tests." ]

[ "Acute gastroenteritis is one of the main causes of mortality in humans and young animals. Domestic and mainly wild animals such as bats, small rodents and birds are highly diversified animals in relation to their habitats and ecological niches and are widely distributed geographically in environments of forest fragmentation in some areas of the Amazon, being considered important sources for viruses that affect humans and other animals. Due to the anthropical activities, these animals changed their natural habitat and adapted to urbanized environments, thus representing risks to human and animal health.", "Due to the anthropical activities, these animals changed their natural habitat and adapted to urbanized environments, thus representing risks to human and animal health. Although the knowledge of the global diversity of enteric viruses is scarce, there are reports demonstrating the detection of rotavirus in domestic animals and animals of productive systems, such as bovines and pigs. The present study investigated the prevalence of Rotavirus A in 648 fecal samples of different animal species from the northeastern mesoregion of the state of Pará, Brazil, which is characterized as an urbanized area with forest fragments.", "The present study investigated the prevalence of Rotavirus A in 648 fecal samples of different animal species from the northeastern mesoregion of the state of Pará, Brazil, which is characterized as an urbanized area with forest fragments. The fecal specimens were collected from October 2014 to April 2016 and subjected to a Qualitative Real-Time Polymerase Chain Reaction (RT-qPCR), using the NSP3 gene as a target. It was observed that 27.5% (178/648) of the samples presented positive results for RVA, with 178 samples distributed in birds (23.6%), canines (21.35%), chiropterans (17.98%), bovines (14.6%), horses (8.43%), small rodents (6.74%), pigs (3.93%) and felines (3.37%), demonstrating the circulation of RVA in domestic animals and suggesting that such proximity could cause transmissions between different species and the occurrence of rearrangements in the genome of RVA as already described in the literature, associated to the traces of environmental degradation in the studied areas.", "It was observed that 27.5% (178/648) of the samples presented positive results for RVA, with 178 samples distributed in birds (23.6%), canines (21.35%), chiropterans (17.98%), bovines (14.6%), horses (8.43%), small rodents (6.74%), pigs (3.93%) and felines (3.37%), demonstrating the circulation of RVA in domestic animals and suggesting that such proximity could cause transmissions between different species and the occurrence of rearrangements in the genome of RVA as already described in the literature, associated to the traces of environmental degradation in the studied areas. Text: Emerging and reemerging infectious diseases are increasing each year in several countries, with an impact both on human populations and on domestic and wild animals living in areas with considerable forest remnants [1] . Most of these diseases are of viral origin, suggesting the emergence and reemergence of viruses that are triggered by human activities that modify the environment [2] .", "Most of these diseases are of viral origin, suggesting the emergence and reemergence of viruses that are triggered by human activities that modify the environment [2] . The populations of wild animals that inhabit forest fragments are strategic groups for studies of public health and the transmission of zoonosis, given that they act as indicators in the assistance and intervention in the human populations, aiming at the prevention of outbreaks and epidemics [3] . Acute gastroenteritis can be caused by infection in the gastrointestinal tract, caused by different infectious or parasite agents [4] [5] [6] [7] .", "Acute gastroenteritis can be caused by infection in the gastrointestinal tract, caused by different infectious or parasite agents [4] [5] [6] [7] . They represent one of the main causes of mortality in humans, and in young animals, counting for about 25% of mortality [8] . Rotavirus is widely distributed in animals, which act as sources of rotavirus emergent strains, with these animals acting in the transmission between species and through reassortment leading to the emergence of new strains which have been reported in human infections [9] [10] [11] [12] .", "Rotavirus is widely distributed in animals, which act as sources of rotavirus emergent strains, with these animals acting in the transmission between species and through reassortment leading to the emergence of new strains which have been reported in human infections [9] [10] [11] [12] . The rotavirus (RV) belongs to the Reoviridae family and comprises nine species known as Rotavirus group A to I, with a recent proposal of the J species [13, 14] . Rotavirus A (RVA) is widespread worldwide and predominantly infects humans, bovines and other mammal species, as well as birds [15] .", "Rotavirus A (RVA) is widespread worldwide and predominantly infects humans, bovines and other mammal species, as well as birds [15] . They have a double-stranded ribonucleic acid (dsRNA) genome, divided into 11 segments coding for structural proteins (VP1-VP4, VP6 and VP7) and nonstructural (NSP1-NSP5/NSP6) proteins [16, 17] . There are records of a close relationship between Amazonian wildlife and human populations [18] , and this interaction is the effect of anthropogenic urbanization activities that result in the deforestation of forest areas, causing the degradation of previously isolated sites such as caves and small caves, a continuous and nature progressive process that has led not only to changes in wildlife habitats but also to a greater relationship with human populations in rural and urban environments, contributing to the occurrence and emergence of diseases different from what normally occurs in endemic regions [19] [20] [21] [22] .", "There are records of a close relationship between Amazonian wildlife and human populations [18] , and this interaction is the effect of anthropogenic urbanization activities that result in the deforestation of forest areas, causing the degradation of previously isolated sites such as caves and small caves, a continuous and nature progressive process that has led not only to changes in wildlife habitats but also to a greater relationship with human populations in rural and urban environments, contributing to the occurrence and emergence of diseases different from what normally occurs in endemic regions [19] [20] [21] [22] . Although the results of RVA have already been described globally [12, [23] [24] [25] [26] [27] [28] [29] [30] , in Brazil, the occurrence, diversity and role of rotavirus in these animals are still poorly studied, considering the large number of present species [4, [31] [32] [33] [34] . In the Brazilian Amazon, especially in the state of Pará, the city of Belém and Northeast metropolitan mesoregions are some of the areas with the highest indexes of environmental changes [35] , which are concentrated, along with the fact that the knowledge of the global diversity of enteric virus in animals is scarce [36] .", "In the Brazilian Amazon, especially in the state of Pará, the city of Belém and Northeast metropolitan mesoregions are some of the areas with the highest indexes of environmental changes [35] , which are concentrated, along with the fact that the knowledge of the global diversity of enteric virus in animals is scarce [36] . Therefore, it is important to monitor the health of domestic and wild animals in their natural habitat, especially in areas with anthropic alterations that have an interface with rural communities and enterprises, in order to investigate the occurrence of RVA in this population. These communities are ecologically complex, because they have multiple hosts and endless pathogens that may eventually circulate in contiguous urban centers, in addition to the fact that it should also be considered that there is still a lack of studies showing the significance of these viruses infecting this population, as in the context of epidemiological surveillance, these animals become important, since they can be considered as natural sources, with the possibility of transmission to humans [37] [38] [39] .", "These communities are ecologically complex, because they have multiple hosts and endless pathogens that may eventually circulate in contiguous urban centers, in addition to the fact that it should also be considered that there is still a lack of studies showing the significance of these viruses infecting this population, as in the context of epidemiological surveillance, these animals become important, since they can be considered as natural sources, with the possibility of transmission to humans [37] [38] [39] . The qualitative real-time polymerase chain reaction (qRT-PCR) used the NSP3 gene and the TaqMan probe from a highly conserved region of the rotavirus non-structural protein 3 (NSP3), which was previously used in samples from human origin and with low viral loads \n\nPrecipitation data were obtained from The Brazilian National Institute of Meteorology (Inmet for the years of capture in the Expedito Ribeiro Settlement (2014) and Açailândia (2015) of the Data Collection Platforms (PCDs) of Belém, located 50 km from Santa Bárbara do Pará, and Tracuateua, located 50 km from Peixe-Boi and 100 km from Viseu. Garmin GPSMap 64s Global Positioning System (GPS) coordinates were collected in the field.", "Garmin GPSMap 64s Global Positioning System (GPS) coordinates were collected in the field. The municipal boundaries were obtained on the website of the Brazilian Institute of Geography and Statistics (IBGE) ( and data on deforestation and land use were obtained from the PRODES [43] and TerraClass [44] Projects. PRODES has annual data in digital format since 2000 and TerraClass presents biannual data since 2004.", "PRODES has annual data in digital format since 2000 and TerraClass presents biannual data since 2004. The satellite image was generated using the sensor Sentinel 2 of the European Space Agency (ESA) ( web/sentinel/user-guides/sentinel-2-msi) with Open Access CC-BY License ( from the years of 2017 and 2018. All the data obtained was stored in a Geographic Database (BDG).", "All the data obtained was stored in a Geographic Database (BDG). The BDG was imported/ stored in a GIS for the editing of the graphic elements, establishment of topological relations between the elements and their respective attributes, spatial analysis and visualization of the result through thematic maps. For the present study, forest fragments of similar size, shape and Phyto physiology were chosen, considering an open peri urban matrix with similar soil use.", "For the present study, forest fragments of similar size, shape and Phyto physiology were chosen, considering an open peri urban matrix with similar soil use. The selected fragments were distributed within the mesoregions studied, and in each selected fragment fecal samples were randomly collected from domestic and wild animals [45] . Soil use classes were obtained from the TerraClass data mosaic from 2004 to 2016, because the study sites were in an area with a high cloud presence, which prevented observation (the area was not observed).", "Soil use classes were obtained from the TerraClass data mosaic from 2004 to 2016, because the study sites were in an area with a high cloud presence, which prevented observation (the area was not observed). The data processing, interpretation, visualization and spatial analysis were performed in ArcGIS software ( For the analysis of data related to the determination of the richness, composition and abundance of the fauna of the animals studied in the study area, considering the collection methods adopted and the species available in each city, each sample was considered as an independent sample. The richness of wild fauna and domestic animals was determined by the total number of species including all collection methods, and the similarity of species was made by the chi-square analysis between the samples of the different treatments with the aid of the EstimateS 8.0 software [46] .", "The richness of wild fauna and domestic animals was determined by the total number of species including all collection methods, and the similarity of species was made by the chi-square analysis between the samples of the different treatments with the aid of the EstimateS 8.0 software [46] . For the calculation of the Test T, the Statistica software was used, and the indices of infected animals in the two environments (forest fragment and peridomicile) were calculated for each treatment sampled by collection area, using the software Past 1.92. Aiming at comparing the values of the diversity indexes through the paired test, as well as the descriptive analysis of the anthropic effects [47] .", "Aiming at comparing the values of the diversity indexes through the paired test, as well as the descriptive analysis of the anthropic effects [47] . The data obtained for the occurrence of RVA and the questionnaires was inserted into a database for a descriptive analysis of the epidemiological profile of the animal population in the three forest ecosystems studied. In this analysis, descriptive statistical treatments were carried out, using customized \"row-columns\" type charts, referring to the data, in order to characterize the sample and quantify the results using absolute frequency values using the chi-square test and the Test T.\n\nPopulation study, collection of clinical specimens and laboratory methodology.", "In this analysis, descriptive statistical treatments were carried out, using customized \"row-columns\" type charts, referring to the data, in order to characterize the sample and quantify the results using absolute frequency values using the chi-square test and the Test T.\n\nPopulation study, collection of clinical specimens and laboratory methodology. The flying animals (wild birds and chiroptera) were captured using mist nets which were opened at dawn (4:00 a.m.) and closed in the morning (9:00 a.m.) and were inspected every one hour until the closing, with a sampling effort of 15 days. This research was approved by National All procedures with animals were performed by veterinarians, being birds and bats identified and released at the same capture site.", "This research was approved by National All procedures with animals were performed by veterinarians, being birds and bats identified and released at the same capture site. The fecal specimens were collected by stimulation of the rectal ampulla with the use of a \"Zaragatoa\", packed in cryogenic vials, identified, stored in liquid nitrogen, and later sent to the Laboratory. Wild animals (small non-flying mammals) were trapped within live-containment traps of the Tomahawk cage (size 45x16x16cm) and Sherman type aluminum (size 30x9x8cm).", "Wild animals (small non-flying mammals) were trapped within live-containment traps of the Tomahawk cage (size 45x16x16cm) and Sherman type aluminum (size 30x9x8cm). In each sample plot, 61 traps were distributed, 20 Shermans and 41 Tomahawks being baited with a mixture made with peanut paste, sardines, cod liver oil and corn meal, as well as fruit like banana, apple and pineapple. All the traps used were inspected daily in the morning, the baits being exchanged when necessary and later after the capture in bags of cloth and at least five specimens of each species were chosen for the collection of biological material.", "All the traps used were inspected daily in the morning, the baits being exchanged when necessary and later after the capture in bags of cloth and at least five specimens of each species were chosen for the collection of biological material. The wild animals were sedated with a combination of ketamine 20mg/kg and xylazine 2mg/kg intramuscularly and subsequently, euthanized with anesthetic overdose of 2% lidocaine in the foramen magnum, according to the recommendation of the National Council for the Control of Animal Experimentation (CONCEA). From October 2014 to April 2016, 1,282 fecal samples were collected from wild and domestic animals.", "From October 2014 to April 2016, 1,282 fecal samples were collected from wild and domestic animals. Amongthese, 648 (50.5%) samples were randomly selected for RVA research and handled in Level Three Biosafety Laboratory (NB3). The viral genome was extracted using the TRIZOL LS REAGENT protocol (INVITRO-GEN, USA/KIT QIAGEN), following the manufacturer's recommendations, with minor adaptation according to the protocol described in the supplemental data.", "The viral genome was extracted using the TRIZOL LS REAGENT protocol (INVITRO-GEN, USA/KIT QIAGEN), following the manufacturer's recommendations, with minor adaptation according to the protocol described in the supplemental data. The qRT-PCR was conducted according to Zeng et al. [40] for the detection of RVA using the NSP3 segment of RVA as the target gene sequence.", "[40] for the detection of RVA using the NSP3 segment of RVA as the target gene sequence. The assay was conducted in a mixture containing: RNAse-free H 2 O, TaqMan RT-PCR Mix (2x), TAqMan RT Enzyme Mix (40x), primers for the NSP3 gene, Primer NSP3 Forward (20mM), Primer NSP3 Reverse (20mM), probe NSP3 S (10nm), Template (RNA) 3μL, having a total reaction volume of 17μL and reverse transcription cycling of 50˚C, 30 minutes, denaturation of 95˚C, 10 minutes, annealing of 45 cycles of 95˚C, 15 seconds and extension of 60˚C, 1 minute. The analyzes were considered positive when presenting the cycle threshold (CT) � 40.", "The analyzes were considered positive when presenting the cycle threshold (CT) � 40. In order to guarantee a reliable test result, the measurements of contamination control were performed with the use of positive animal control (SA11 prototype) and a negative control (ultrapure water). All RVA-positive samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) according to Mijatovic et al [41] to genotyping low viral loads samples.", "All RVA-positive samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) according to Mijatovic et al [41] to genotyping low viral loads samples. First round was performed with consensus primers N-VP4F1/N-VP4R1 and the Nested-PCR was conducted with N-VP4F2/N-VP4R2 primers to amplify VP4 gene. Amplicons were purified and sequencing for VP4 gene using the same primers of Nested-PCR.", "Amplicons were purified and sequencing for VP4 gene using the same primers of Nested-PCR. The sequences were collected from an automated ABI Prism 3130xl DNA sequencer (Applied Biosystems). The sequence fragments were assembled and edited using the Geneious Bioinformatics software platform v.8.1.7.", "The sequence fragments were assembled and edited using the Geneious Bioinformatics software platform v.8.1.7. Posteriorly, the data were compared with othersequences from the National Center for Biotechnology Information GenBank database using BLAST alignment tool to elucidate the RVA genotype of the samples. From October 2014 to April 2016, a total of 648 fecal samples of wild and domestic animals belonging to three forest fragments areas were tested for the NSP3 gene by qualitative qPCR, and 178 (27.5%) were positive for RVA, distributed among the species: birds (23.6%), canines (21.35%), bats (17.98%), cattle (14.6%), horses (8.43%), small rodents (6.74%), swine (3.93%) and felines (3.37%).", "From October 2014 to April 2016, a total of 648 fecal samples of wild and domestic animals belonging to three forest fragments areas were tested for the NSP3 gene by qualitative qPCR, and 178 (27.5%) were positive for RVA, distributed among the species: birds (23.6%), canines (21.35%), bats (17.98%), cattle (14.6%), horses (8.43%), small rodents (6.74%), swine (3.93%) and felines (3.37%). The CT interval ranged from 28. 47 It was possible to detect viral strains in all genders of animals studied and in the harvesting period none of the animals showed signs of acute infection and / or diarrhea.", "47 It was possible to detect viral strains in all genders of animals studied and in the harvesting period none of the animals showed signs of acute infection and / or diarrhea. Rotavirus A (RVA) detected in the present study of wild and domestic animals belonging to the three areas of forest fragment, according to Fig 2. In relation to the evaluated bovines, only in the city of Viseu, these species were studied because they were created extensively.", "In relation to the evaluated bovines, only in the city of Viseu, these species were studied because they were created extensively. In addition, most of the animals were young with ages varying from 1 day to 8 yearsold, history of deficient vaccination, lack of technical assistance and raised in the form of subsistence. The animals showed no symptoms of diarrhea, only low weight performance and poor sanitary management status.", "The animals showed no symptoms of diarrhea, only low weight performance and poor sanitary management status. In relation to chiroptera, 32 (17.98%) positive samples for RVA were distributed among Carollia perspicillata species, with 12 (37.5%) being all adults, 9 (28.12%) Desmodus rotundus samples (4 young and 5 adults), 5 (15.6%) of Uroderma bilobata (15.62%), 3 (9.37%) of Artibeus lituratus and the species Artibeus Planirostus, Diaemus iyoug and Glossophagine with 1 (3.12%) each. These animals came from areas of forest fragments located near bovine and equine farms, in addition to inhabiting small chicken farms.", "These animals came from areas of forest fragments located near bovine and equine farms, in addition to inhabiting small chicken farms. Fig 3 shows the results obtained for all the species of animals investigated in the forest fragment as well as in the peridomicillus area. The anthropic variables were analyzed for the three cities studied, as well as the use of the soil within the range of the animals, obeying the domicile, the peridomicile and the forest fragment where the traps of small rodents, birds and various species of animals were captured (Fig 4 and Fig 5) .", "The anthropic variables were analyzed for the three cities studied, as well as the use of the soil within the range of the animals, obeying the domicile, the peridomicile and the forest fragment where the traps of small rodents, birds and various species of animals were captured (Fig 4 and Fig 5) . Considering the factors related to the anthropic activities in the three studied areas within the three cities of the present study, it was observed that the city of Santa Bárbara is the one that has a better area of preserved forest and the city of Viseu a smaller area. However, in the city of Santa Bárbara, a greater concentration of occupations was observed around the area of forest fragment.", "However, in the city of Santa Bárbara, a greater concentration of occupations was observed around the area of forest fragment. It was observed in this chosen area of the city, the presence of different families living in a rural settlement, surviving from the exploitation of forest resources and the creation of small animals for subsistence, such as poultry and fish farming, as well as family farming products. The breeding of animals in native pastures was only observed in the cities of Peixe Boi and Viseu.", "The breeding of animals in native pastures was only observed in the cities of Peixe Boi and Viseu. Extensive livestock farming was practiced with beef cattle, equines for work and small animals (swine and goats). In relation to the most preserved pasture area, the city of Peixe Boi had the largest area, according to the data shown in Fig 5, however, in the city of Viseu, a higher regeneration was observed in the pastures during the period of the study, with significant secondary vegetation.", "In relation to the most preserved pasture area, the city of Peixe Boi had the largest area, according to the data shown in Fig 5, however, in the city of Viseu, a higher regeneration was observed in the pastures during the period of the study, with significant secondary vegetation. When comparing the climates of the three areas it was observed that the predominant climate is megathermal and humid with average annual temperature around 27˚C. The months of October, November and December are the hottest, with temperatures between 32˚C and 34˚C and absolute maximums around 41˚C.", "The months of October, November and December are the hottest, with temperatures between 32˚C and 34˚C and absolute maximums around 41˚C. Annual rainfall is quite high, generally around 2,350 mm, but strongly concentrated from January to June (80%). From September to December, on the contrary, rainfall is rare, about 7%, with a short dry season, of moderate water deficit in those months.", "From September to December, on the contrary, rainfall is rare, about 7%, with a short dry season, of moderate water deficit in those months. The relative humidity of the average air oscillates around 85%, as shown in Fig 6 [48] . The description of the accumulated precipitation in the year of capture of the fecal specimens compared to the Climatological Normals (CLINO) for the period from 1961-1990 of the PCDs closest to the locations of the Expedito Ribeiro / Santa Bárbara settlement (Belém PCD), Vila Ananim / Peixe-Boi and Açaiteua / Viseu (Tracauateua PCD) show the frequency of rainfall in the regions, which facilitates the renewal of the pastures and the regeneration of the impacted forests, being an important indicator of the reduction of the damages caused by deforestation in the region.", "The description of the accumulated precipitation in the year of capture of the fecal specimens compared to the Climatological Normals (CLINO) for the period from 1961-1990 of the PCDs closest to the locations of the Expedito Ribeiro / Santa Bárbara settlement (Belém PCD), Vila Ananim / Peixe-Boi and Açaiteua / Viseu (Tracauateua PCD) show the frequency of rainfall in the regions, which facilitates the renewal of the pastures and the regeneration of the impacted forests, being an important indicator of the reduction of the damages caused by deforestation in the region. The average deforestation index in the three study areas was calculated from data obtained from INPE information systems. It was observed that in the years of 2013 to 2014 there were no changes in these regions; in the period from 2014 to 2015 about 4.1% of the city of Viseu was changed and 1.6% of the city of Peixe Boi.", "It was observed that in the years of 2013 to 2014 there were no changes in these regions; in the period from 2014 to 2015 about 4.1% of the city of Viseu was changed and 1.6% of the city of Peixe Boi. In relation to the period of 2016, great changes were observed in Peixe Boi (79%) and in the city of Viseu (70%), thus demonstrating that changes in the natural ecosystem may be associated with the frequencies for RVA in the studied areas, according to Fig 7. When assessing the infected animals in relation to the uninfected animals in both the forest fragment and the peridomicile, considering as animals of the forest fragment the birds, the chiroptera and the small rodents and as animals of the peridomicile the canines, bovines, pigs, felines and horses, a percentage of 37.07% infected peri domestic animals (86/232) and 22.12% infected forest fragment animals (92/416) were obtained.", "When assessing the infected animals in relation to the uninfected animals in both the forest fragment and the peridomicile, considering as animals of the forest fragment the birds, the chiroptera and the small rodents and as animals of the peridomicile the canines, bovines, pigs, felines and horses, a percentage of 37.07% infected peri domestic animals (86/232) and 22.12% infected forest fragment animals (92/416) were obtained. Applying the selected statistical analysis, a Pearson x2 Chi-square value was obtained: 16.7159, df = 1 and p <0.001, meaning that the hypothesis was corroborated, that is, the greater the degradation of the environment, the more likely it will be the search for food by wild animals in adjacent areas, or in the edge of the forest or even in the peri domiciliary region. In this sense, the possibility of contagion with other species of animals, even humans, should be considered because of the capacity of the rotavirus to be transmitted via the fecal / oral route or through direct contact with the environment.", "In this sense, the possibility of contagion with other species of animals, even humans, should be considered because of the capacity of the rotavirus to be transmitted via the fecal / oral route or through direct contact with the environment. It is important to point out that the animals detected in this study are important sources of viral strains. A total of 80 stool samples were selected, reextracted and analyzed using PCR for the VP4 gene.", "A total of 80 stool samples were selected, reextracted and analyzed using PCR for the VP4 gene. Eight strains (10%) were positive for VP4 gene, being 2 strains bellowed to P [6] genotype and 6 to P[4]-type, according to \n\nIn the present study, RVA was detected circulating in 27.5% of the animals; 36% in domestic animals and 64% in wild animals, providing a unique dataset with qRT-PCR detecting a low viral load of RVA in different species, which further correlates with the deforestation index. These data are important because there is a lack of tests for RVA diagnosis in animals, since the current methods of RVA detection does not always detect in these populations [8] .", "These data are important because there is a lack of tests for RVA diagnosis in animals, since the current methods of RVA detection does not always detect in these populations [8] . With the advent of real-time PCR (qPCR), there was an exponential growth, compared to conventional PCR essays, since its superior accuracy, sensitivity and specificity is remarkable, and it is Rotavirus A in wild and domestic animals possible to detect RVA in a variety of animal species using NSP3 gene [49] . The sensitivity of RT-qPCR significantly improved the rate of RVA detection in clinical samples from animals and in this context, the present study proposed an interesting study metrics using virus spreading in the wild animals which inhabit forest fragments to indicate human population interventions, with the goal of preventing the virus outbreaks leveraged on the unique geographic characteristics of Brazil and its large number of species in Amazon.", "The sensitivity of RT-qPCR significantly improved the rate of RVA detection in clinical samples from animals and in this context, the present study proposed an interesting study metrics using virus spreading in the wild animals which inhabit forest fragments to indicate human population interventions, with the goal of preventing the virus outbreaks leveraged on the unique geographic characteristics of Brazil and its large number of species in Amazon. Currently, no data have been described in the literature regarding the RVA detection using real-time qPCR technique in a wide variety of wild animal species. However, a study by Soltan et al.", "However, a study by Soltan et al. [50] conducted with horses and cattle detected RVA by RT-PCR, commercial RT-PCR and RT-qPCR in 36.7%, 51.4% and 56.9% respectively, differently from the present study that showed higher positivity for chiropterans (17.98%), canines (21.35%), birds (23.6%) and cattle (14.6%). The first description of RVA in chiroptera was recorded in feces of Eidolon helvum caught in Vihiga, Kenya [51] .", "The first description of RVA in chiroptera was recorded in feces of Eidolon helvum caught in Vihiga, Kenya [51] . Afterwards, several strains of RVA were detected by different molecular techniques involving chiroptera, in several countries, including Kenya (E. helvum), China (Rhinolophus hipposideros and Aselliscus stoliczkanus), France (Myotis mystacinus), Cameroon (E. helvum) and Brazil [31, [51] [52] [53] [54] [55] . The present study shows the occurrence of RVA in 17.98% of the chiroptera, being among the species Carollia perspicillata (37.5%), Desmodus rotundus (28.12%), Uroderma bilobata (15.6%), Artibeus lituratus (9.37%), Artibeus Planirostus (3.12%), Diaemus iyoug and Glossophagine (3.12%).", "The present study shows the occurrence of RVA in 17.98% of the chiroptera, being among the species Carollia perspicillata (37.5%), Desmodus rotundus (28.12%), Uroderma bilobata (15.6%), Artibeus lituratus (9.37%), Artibeus Planirostus (3.12%), Diaemus iyoug and Glossophagine (3.12%). Barquez et al. [56] reported that Desmodus rotundus is one of the three hematophagous species of the Phyllostomidae family, found throughout South America, Central America and Mexico.", "[56] reported that Desmodus rotundus is one of the three hematophagous species of the Phyllostomidae family, found throughout South America, Central America and Mexico. Of the positive chiroptera for RVA in the present study, a prevalence of 28.12% was of Desmodus rotundus. This species feeds on birds, can feed on mammals, mostly medium or large, facilitating the dissemination of viral spores among the community within the habitat, as observed in the present study.", "This species feeds on birds, can feed on mammals, mostly medium or large, facilitating the dissemination of viral spores among the community within the habitat, as observed in the present study. These findings show the importance of epidemiological data on the studied species due to the lack of studies involving species of neotropical chiroptera, and it is not possible to establish comparative parameters for these animals. Regarding the circulation of RVA in canines and birds, the prevalence was 53% and 29%, respectively.", "Regarding the circulation of RVA in canines and birds, the prevalence was 53% and 29%, respectively. Although in the Amazon region there are records of RVA, RVD, RVF and RVG that infect birds [57] [58] and RVD in migratory birds [59] , all were detected by RT-PCR assays differently from the present study which detected the RVA by RT-qPCR involving a variety of animal species. On the other hand, the prevalence in felines (16%) and pigs (22%) was lower, probably because there are few animals of these species in the region, as well as few creations.", "On the other hand, the prevalence in felines (16%) and pigs (22%) was lower, probably because there are few animals of these species in the region, as well as few creations. The study detected the presence of RVA in different species of animals both in areas near the home and in areas located in fragments of forest, characterized as forest remnants, since they were located in cities that suffered high environmental impacts due to vegetal extractivism, pasture formation for cattle breeding, exploitation of natural resources, and direct reflexes on the habitats of wild animals that can serve as virus sources, thus facilitating the dispersion of RVA among communities of coexisting animals. It is worth emphasizing that these animals have a greater contact with the human populations of the studied areas since they cohabit with the humans in the region, besides having a high flow of movement between the forest extracts and environments chosen for the present study.", "It is worth emphasizing that these animals have a greater contact with the human populations of the studied areas since they cohabit with the humans in the region, besides having a high flow of movement between the forest extracts and environments chosen for the present study. However, it is noteworthy that only in the communities of Santa Bárbara and Viseu were collected fecal specimens from asymptomatic humans for diarrhea and tested for RVA, but all were negative. It is notorious yet, the existence of different levels of degradation in the studied environments, considering the presented data.", "It is notorious yet, the existence of different levels of degradation in the studied environments, considering the presented data. The fragmentation of the forest generates many consequences on the Amazonian biota, being able to alter the diversity and the composition of the animal communities in the fragments and even to interfere in the ecological processes, without considering that the fragments of forest in the Amazon are influenced by the climate, possibly facilitating the dispersion of pathogens by the environment, since the wild animals detected in the present study are asymptomatic and have low viral load for RVA. The occurrence of RVA in this population of animals may explain the possibility of dispersion of viral strains, since there is a proximity to the human population, besides the biological characteristics of these species that may represent important sources for gastroenteric viruses, along with the fact that all animals were asymptomatic for diarrhea.", "The occurrence of RVA in this population of animals may explain the possibility of dispersion of viral strains, since there is a proximity to the human population, besides the biological characteristics of these species that may represent important sources for gastroenteric viruses, along with the fact that all animals were asymptomatic for diarrhea. Wild birds have unlimited flight capacity, were captured in an interface region between the peridomicile and forest fragments and it is believed that this region has not been influenced by anthropic activities such as those observed in the area of the present study. On the other hand, the breeding method for poultry and canines close to homes and the forest ecosystem, as they are created in the communities surveyed, probably facilitates direct contact with possible sources of contamination, since in the areas the use of septic tanks is deficient and sometimes non-existent, which may facilitate or even increase the risk of viral dispersion throughout the environment.", "On the other hand, the breeding method for poultry and canines close to homes and the forest ecosystem, as they are created in the communities surveyed, probably facilitates direct contact with possible sources of contamination, since in the areas the use of septic tanks is deficient and sometimes non-existent, which may facilitate or even increase the risk of viral dispersion throughout the environment. The high rates of increase and the analysis of land use in the researched areas may be important indicators of how these animals interact, since with deforestation, the populations of wild animals seek refuge in nearby communities facilitating the dispersal of infectious agents and the possible occurrence of carrier animals by direct contact or contamination of the local environment. To our knowledge, this is the first study in which a real-time PCR assay was applied for the detection of RVA involving a wide variety of domestic and wild animals, facilitating practical utility in epidemiological and molecular studies and assisting in a perspective in the elaboration of sanitary control and monitoring, preventing possible outbreaks in the studied communities.", "To our knowledge, this is the first study in which a real-time PCR assay was applied for the detection of RVA involving a wide variety of domestic and wild animals, facilitating practical utility in epidemiological and molecular studies and assisting in a perspective in the elaboration of sanitary control and monitoring, preventing possible outbreaks in the studied communities. The detection of positive animals was useful to monitor the infection of the agent in the animal population and to provide an early warning signal to predict an impending epidemic and a favorable risk for the human population, given the evidence of RVA circulation in the different forest fragments. In addition, the RT-qPCR assay may be a useful alternative for the differential diagnosis of RV in possible coexisting mixed infections clinically indistinguishable such as those caused by other viral strains that cause gastroenteritis such as: astrovirus, coronavirus, picobirnavirus, calicivirus, among others as observed in the studies of Jing et al.", "In addition, the RT-qPCR assay may be a useful alternative for the differential diagnosis of RV in possible coexisting mixed infections clinically indistinguishable such as those caused by other viral strains that cause gastroenteritis such as: astrovirus, coronavirus, picobirnavirus, calicivirus, among others as observed in the studies of Jing et al. [60] and Waruhiu et al. [61] .", "[60] and Waruhiu et al. [61] . Diarrhea associated with RVA infections in pigs is an important cause of increased mortality and economic losses in Europe.", "Diarrhea associated with RVA infections in pigs is an important cause of increased mortality and economic losses in Europe. The most prevalent genotypes isolated from feces of Belgian diarrheal and non-diarrheal piglets in 2012 [62] demonstrate a wide range of combinations of genotypes G / P including; G3P [6] , G4P [6] , G5P [6] , G4P [7] , G5P [7] , G9P [7] , G9P [13] and G9P [23] . On the other hand, in the present study it was possible to detect only P [6] genotype, since majority of samples was asymptomatic for diarrhea.", "On the other hand, in the present study it was possible to detect only P [6] genotype, since majority of samples was asymptomatic for diarrhea. Finding shows that different P genotypes of RVA strains interact with distinct blood group histological antigens (HBGA, ABOH, Lewis) and sialic acids via VP4 providing insight into the regional prevalence and increased zoonotic potential of some RVA of origin swine [63] . The genotype P [6] was identified in piglets in Brazil [64] and in Italy and Japan resembling genotype P [6] human [65, 66] .", "The genotype P [6] was identified in piglets in Brazil [64] and in Italy and Japan resembling genotype P [6] human [65, 66] . In the population of animals studied the zoonotic transmission can be frequent, since the animals live in contact with humans and in precarious sanitary conditions. In Brazil, this genotype was described in animal and human populations in studies of Luchs et al.", "In Brazil, this genotype was described in animal and human populations in studies of Luchs et al. [32] ; Honma et al. [67] ; Araújo et al. [68] ; Mascarenhas et al. [69] and Lorenzetti et al.", "[68] ; Mascarenhas et al. [69] and Lorenzetti et al. [70] such studies corroborate the importance of continuing to monitor genotypes to verify if uncommon strains or new strains are emerging and can infect animal populations or inter-species transmissions.", "[70] such studies corroborate the importance of continuing to monitor genotypes to verify if uncommon strains or new strains are emerging and can infect animal populations or inter-species transmissions. Regarding the genotype P [4] , itwas most detected in our samples in bats, dogs, swine and feline. This genotype is not common in animals, being more detected in human and environmental samples in various parts of the world and included our region [71] .", "This genotype is not common in animals, being more detected in human and environmental samples in various parts of the world and included our region [71] . It is important to emphasize that the indicators of environmental contamination in Brazil are significant and contribute to the possibility of human-animal transmission [71] . Such data need further investigation in later work to better characterize the interspecies transmission, since the occurrence of enteric viruses in different matrices demonstrates the anthropogenic impact of the exposed population around and points to the potential risk of infection by the possible exposure of individuals susceptible.", "Such data need further investigation in later work to better characterize the interspecies transmission, since the occurrence of enteric viruses in different matrices demonstrates the anthropogenic impact of the exposed population around and points to the potential risk of infection by the possible exposure of individuals susceptible. Our findings may be useful for tracking fecal contamination in the environment using animals as possible sources thus minimizing the risk of infection by exposure to susceptible individuals, in this case different animal species or even human populations. RVA were detected in wild and domestic animals using a RT-qPCR assay that analyzed samples that had low viral load for RVA.", "RVA were detected in wild and domestic animals using a RT-qPCR assay that analyzed samples that had low viral load for RVA. Although the samples are asymptomatic for diarrhea, it is necessary to conduct strategies for the monitoring and control of the animals in the areas studied in the human population as well as in other species of animals, as well as the implementation of preventive measures aimed at future outbreaks in communities animals in the resident population in these impacted areas. Therefore, the present study is unprecedented in the region and in the country in relation to the research of RVA in wild animals.", "Therefore, the present study is unprecedented in the region and in the country in relation to the research of RVA in wild animals. It is noteworthy that, although the quality of the analyzed samples is characterized as low detectable viral load, the technique presented a good analytical response in the detection of the source animals for RVA, facilitating the selection of the samples for future genetic characterization tests." ]

[ "Acute gastroenteritis is one of the main causes of mortality in humans and young animals. Domestic and mainly wild animals such as bats, small rodents and birds are highly diversified animals in relation to their habitats and ecological niches and are widely distributed geographically in environments of forest fragmentation in some areas of the Amazon, being considered important sources for viruses that affect humans and other animals. Due to the anthropical activities, these animals changed their natural habitat and adapted to urbanized environments, thus representing risks to human and animal health.", "Due to the anthropical activities, these animals changed their natural habitat and adapted to urbanized environments, thus representing risks to human and animal health. Although the knowledge of the global diversity of enteric viruses is scarce, there are reports demonstrating the detection of rotavirus in domestic animals and animals of productive systems, such as bovines and pigs. The present study investigated the prevalence of Rotavirus A in 648 fecal samples of different animal species from the northeastern mesoregion of the state of Pará, Brazil, which is characterized as an urbanized area with forest fragments.", "The present study investigated the prevalence of Rotavirus A in 648 fecal samples of different animal species from the northeastern mesoregion of the state of Pará, Brazil, which is characterized as an urbanized area with forest fragments. The fecal specimens were collected from October 2014 to April 2016 and subjected to a Qualitative Real-Time Polymerase Chain Reaction (RT-qPCR), using the NSP3 gene as a target. It was observed that 27.5% (178/648) of the samples presented positive results for RVA, with 178 samples distributed in birds (23.6%), canines (21.35%), chiropterans (17.98%), bovines (14.6%), horses (8.43%), small rodents (6.74%), pigs (3.93%) and felines (3.37%), demonstrating the circulation of RVA in domestic animals and suggesting that such proximity could cause transmissions between different species and the occurrence of rearrangements in the genome of RVA as already described in the literature, associated to the traces of environmental degradation in the studied areas.", "It was observed that 27.5% (178/648) of the samples presented positive results for RVA, with 178 samples distributed in birds (23.6%), canines (21.35%), chiropterans (17.98%), bovines (14.6%), horses (8.43%), small rodents (6.74%), pigs (3.93%) and felines (3.37%), demonstrating the circulation of RVA in domestic animals and suggesting that such proximity could cause transmissions between different species and the occurrence of rearrangements in the genome of RVA as already described in the literature, associated to the traces of environmental degradation in the studied areas. Text: Emerging and reemerging infectious diseases are increasing each year in several countries, with an impact both on human populations and on domestic and wild animals living in areas with considerable forest remnants [1] . Most of these diseases are of viral origin, suggesting the emergence and reemergence of viruses that are triggered by human activities that modify the environment [2] .", "Most of these diseases are of viral origin, suggesting the emergence and reemergence of viruses that are triggered by human activities that modify the environment [2] . The populations of wild animals that inhabit forest fragments are strategic groups for studies of public health and the transmission of zoonosis, given that they act as indicators in the assistance and intervention in the human populations, aiming at the prevention of outbreaks and epidemics [3] . Acute gastroenteritis can be caused by infection in the gastrointestinal tract, caused by different infectious or parasite agents [4] [5] [6] [7] .", "Acute gastroenteritis can be caused by infection in the gastrointestinal tract, caused by different infectious or parasite agents [4] [5] [6] [7] . They represent one of the main causes of mortality in humans, and in young animals, counting for about 25% of mortality [8] . Rotavirus is widely distributed in animals, which act as sources of rotavirus emergent strains, with these animals acting in the transmission between species and through reassortment leading to the emergence of new strains which have been reported in human infections [9] [10] [11] [12] .", "Rotavirus is widely distributed in animals, which act as sources of rotavirus emergent strains, with these animals acting in the transmission between species and through reassortment leading to the emergence of new strains which have been reported in human infections [9] [10] [11] [12] . The rotavirus (RV) belongs to the Reoviridae family and comprises nine species known as Rotavirus group A to I, with a recent proposal of the J species [13, 14] . Rotavirus A (RVA) is widespread worldwide and predominantly infects humans, bovines and other mammal species, as well as birds [15] .", "Rotavirus A (RVA) is widespread worldwide and predominantly infects humans, bovines and other mammal species, as well as birds [15] . They have a double-stranded ribonucleic acid (dsRNA) genome, divided into 11 segments coding for structural proteins (VP1-VP4, VP6 and VP7) and nonstructural (NSP1-NSP5/NSP6) proteins [16, 17] . There are records of a close relationship between Amazonian wildlife and human populations [18] , and this interaction is the effect of anthropogenic urbanization activities that result in the deforestation of forest areas, causing the degradation of previously isolated sites such as caves and small caves, a continuous and nature progressive process that has led not only to changes in wildlife habitats but also to a greater relationship with human populations in rural and urban environments, contributing to the occurrence and emergence of diseases different from what normally occurs in endemic regions [19] [20] [21] [22] .", "There are records of a close relationship between Amazonian wildlife and human populations [18] , and this interaction is the effect of anthropogenic urbanization activities that result in the deforestation of forest areas, causing the degradation of previously isolated sites such as caves and small caves, a continuous and nature progressive process that has led not only to changes in wildlife habitats but also to a greater relationship with human populations in rural and urban environments, contributing to the occurrence and emergence of diseases different from what normally occurs in endemic regions [19] [20] [21] [22] . Although the results of RVA have already been described globally [12, [23] [24] [25] [26] [27] [28] [29] [30] , in Brazil, the occurrence, diversity and role of rotavirus in these animals are still poorly studied, considering the large number of present species [4, [31] [32] [33] [34] . In the Brazilian Amazon, especially in the state of Pará, the city of Belém and Northeast metropolitan mesoregions are some of the areas with the highest indexes of environmental changes [35] , which are concentrated, along with the fact that the knowledge of the global diversity of enteric virus in animals is scarce [36] .", "In the Brazilian Amazon, especially in the state of Pará, the city of Belém and Northeast metropolitan mesoregions are some of the areas with the highest indexes of environmental changes [35] , which are concentrated, along with the fact that the knowledge of the global diversity of enteric virus in animals is scarce [36] . Therefore, it is important to monitor the health of domestic and wild animals in their natural habitat, especially in areas with anthropic alterations that have an interface with rural communities and enterprises, in order to investigate the occurrence of RVA in this population. These communities are ecologically complex, because they have multiple hosts and endless pathogens that may eventually circulate in contiguous urban centers, in addition to the fact that it should also be considered that there is still a lack of studies showing the significance of these viruses infecting this population, as in the context of epidemiological surveillance, these animals become important, since they can be considered as natural sources, with the possibility of transmission to humans [37] [38] [39] .", "These communities are ecologically complex, because they have multiple hosts and endless pathogens that may eventually circulate in contiguous urban centers, in addition to the fact that it should also be considered that there is still a lack of studies showing the significance of these viruses infecting this population, as in the context of epidemiological surveillance, these animals become important, since they can be considered as natural sources, with the possibility of transmission to humans [37] [38] [39] . The qualitative real-time polymerase chain reaction (qRT-PCR) used the NSP3 gene and the TaqMan probe from a highly conserved region of the rotavirus non-structural protein 3 (NSP3), which was previously used in samples from human origin and with low viral loads \n\nPrecipitation data were obtained from The Brazilian National Institute of Meteorology (Inmet for the years of capture in the Expedito Ribeiro Settlement (2014) and Açailândia (2015) of the Data Collection Platforms (PCDs) of Belém, located 50 km from Santa Bárbara do Pará, and Tracuateua, located 50 km from Peixe-Boi and 100 km from Viseu. Garmin GPSMap 64s Global Positioning System (GPS) coordinates were collected in the field.", "Garmin GPSMap 64s Global Positioning System (GPS) coordinates were collected in the field. The municipal boundaries were obtained on the website of the Brazilian Institute of Geography and Statistics (IBGE) ( and data on deforestation and land use were obtained from the PRODES [43] and TerraClass [44] Projects. PRODES has annual data in digital format since 2000 and TerraClass presents biannual data since 2004.", "PRODES has annual data in digital format since 2000 and TerraClass presents biannual data since 2004. The satellite image was generated using the sensor Sentinel 2 of the European Space Agency (ESA) ( web/sentinel/user-guides/sentinel-2-msi) with Open Access CC-BY License ( from the years of 2017 and 2018. All the data obtained was stored in a Geographic Database (BDG).", "All the data obtained was stored in a Geographic Database (BDG). The BDG was imported/ stored in a GIS for the editing of the graphic elements, establishment of topological relations between the elements and their respective attributes, spatial analysis and visualization of the result through thematic maps. For the present study, forest fragments of similar size, shape and Phyto physiology were chosen, considering an open peri urban matrix with similar soil use.", "For the present study, forest fragments of similar size, shape and Phyto physiology were chosen, considering an open peri urban matrix with similar soil use. The selected fragments were distributed within the mesoregions studied, and in each selected fragment fecal samples were randomly collected from domestic and wild animals [45] . Soil use classes were obtained from the TerraClass data mosaic from 2004 to 2016, because the study sites were in an area with a high cloud presence, which prevented observation (the area was not observed).", "Soil use classes were obtained from the TerraClass data mosaic from 2004 to 2016, because the study sites were in an area with a high cloud presence, which prevented observation (the area was not observed). The data processing, interpretation, visualization and spatial analysis were performed in ArcGIS software ( For the analysis of data related to the determination of the richness, composition and abundance of the fauna of the animals studied in the study area, considering the collection methods adopted and the species available in each city, each sample was considered as an independent sample. The richness of wild fauna and domestic animals was determined by the total number of species including all collection methods, and the similarity of species was made by the chi-square analysis between the samples of the different treatments with the aid of the EstimateS 8.0 software [46] .", "The richness of wild fauna and domestic animals was determined by the total number of species including all collection methods, and the similarity of species was made by the chi-square analysis between the samples of the different treatments with the aid of the EstimateS 8.0 software [46] . For the calculation of the Test T, the Statistica software was used, and the indices of infected animals in the two environments (forest fragment and peridomicile) were calculated for each treatment sampled by collection area, using the software Past 1.92. Aiming at comparing the values of the diversity indexes through the paired test, as well as the descriptive analysis of the anthropic effects [47] .", "Aiming at comparing the values of the diversity indexes through the paired test, as well as the descriptive analysis of the anthropic effects [47] . The data obtained for the occurrence of RVA and the questionnaires was inserted into a database for a descriptive analysis of the epidemiological profile of the animal population in the three forest ecosystems studied. In this analysis, descriptive statistical treatments were carried out, using customized \"row-columns\" type charts, referring to the data, in order to characterize the sample and quantify the results using absolute frequency values using the chi-square test and the Test T.\n\nPopulation study, collection of clinical specimens and laboratory methodology.", "In this analysis, descriptive statistical treatments were carried out, using customized \"row-columns\" type charts, referring to the data, in order to characterize the sample and quantify the results using absolute frequency values using the chi-square test and the Test T.\n\nPopulation study, collection of clinical specimens and laboratory methodology. The flying animals (wild birds and chiroptera) were captured using mist nets which were opened at dawn (4:00 a.m.) and closed in the morning (9:00 a.m.) and were inspected every one hour until the closing, with a sampling effort of 15 days. This research was approved by National All procedures with animals were performed by veterinarians, being birds and bats identified and released at the same capture site.", "This research was approved by National All procedures with animals were performed by veterinarians, being birds and bats identified and released at the same capture site. The fecal specimens were collected by stimulation of the rectal ampulla with the use of a \"Zaragatoa\", packed in cryogenic vials, identified, stored in liquid nitrogen, and later sent to the Laboratory. Wild animals (small non-flying mammals) were trapped within live-containment traps of the Tomahawk cage (size 45x16x16cm) and Sherman type aluminum (size 30x9x8cm).", "Wild animals (small non-flying mammals) were trapped within live-containment traps of the Tomahawk cage (size 45x16x16cm) and Sherman type aluminum (size 30x9x8cm). In each sample plot, 61 traps were distributed, 20 Shermans and 41 Tomahawks being baited with a mixture made with peanut paste, sardines, cod liver oil and corn meal, as well as fruit like banana, apple and pineapple. All the traps used were inspected daily in the morning, the baits being exchanged when necessary and later after the capture in bags of cloth and at least five specimens of each species were chosen for the collection of biological material.", "All the traps used were inspected daily in the morning, the baits being exchanged when necessary and later after the capture in bags of cloth and at least five specimens of each species were chosen for the collection of biological material. The wild animals were sedated with a combination of ketamine 20mg/kg and xylazine 2mg/kg intramuscularly and subsequently, euthanized with anesthetic overdose of 2% lidocaine in the foramen magnum, according to the recommendation of the National Council for the Control of Animal Experimentation (CONCEA). From October 2014 to April 2016, 1,282 fecal samples were collected from wild and domestic animals.", "From October 2014 to April 2016, 1,282 fecal samples were collected from wild and domestic animals. Amongthese, 648 (50.5%) samples were randomly selected for RVA research and handled in Level Three Biosafety Laboratory (NB3). The viral genome was extracted using the TRIZOL LS REAGENT protocol (INVITRO-GEN, USA/KIT QIAGEN), following the manufacturer's recommendations, with minor adaptation according to the protocol described in the supplemental data.", "The viral genome was extracted using the TRIZOL LS REAGENT protocol (INVITRO-GEN, USA/KIT QIAGEN), following the manufacturer's recommendations, with minor adaptation according to the protocol described in the supplemental data. The qRT-PCR was conducted according to Zeng et al. [40] for the detection of RVA using the NSP3 segment of RVA as the target gene sequence.", "[40] for the detection of RVA using the NSP3 segment of RVA as the target gene sequence. The assay was conducted in a mixture containing: RNAse-free H 2 O, TaqMan RT-PCR Mix (2x), TAqMan RT Enzyme Mix (40x), primers for the NSP3 gene, Primer NSP3 Forward (20mM), Primer NSP3 Reverse (20mM), probe NSP3 S (10nm), Template (RNA) 3μL, having a total reaction volume of 17μL and reverse transcription cycling of 50˚C, 30 minutes, denaturation of 95˚C, 10 minutes, annealing of 45 cycles of 95˚C, 15 seconds and extension of 60˚C, 1 minute. The analyzes were considered positive when presenting the cycle threshold (CT) � 40.", "The analyzes were considered positive when presenting the cycle threshold (CT) � 40. In order to guarantee a reliable test result, the measurements of contamination control were performed with the use of positive animal control (SA11 prototype) and a negative control (ultrapure water). All RVA-positive samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) according to Mijatovic et al [41] to genotyping low viral loads samples.", "All RVA-positive samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) according to Mijatovic et al [41] to genotyping low viral loads samples. First round was performed with consensus primers N-VP4F1/N-VP4R1 and the Nested-PCR was conducted with N-VP4F2/N-VP4R2 primers to amplify VP4 gene. Amplicons were purified and sequencing for VP4 gene using the same primers of Nested-PCR.", "Amplicons were purified and sequencing for VP4 gene using the same primers of Nested-PCR. The sequences were collected from an automated ABI Prism 3130xl DNA sequencer (Applied Biosystems). The sequence fragments were assembled and edited using the Geneious Bioinformatics software platform v.8.1.7.", "The sequence fragments were assembled and edited using the Geneious Bioinformatics software platform v.8.1.7. Posteriorly, the data were compared with othersequences from the National Center for Biotechnology Information GenBank database using BLAST alignment tool to elucidate the RVA genotype of the samples. From October 2014 to April 2016, a total of 648 fecal samples of wild and domestic animals belonging to three forest fragments areas were tested for the NSP3 gene by qualitative qPCR, and 178 (27.5%) were positive for RVA, distributed among the species: birds (23.6%), canines (21.35%), bats (17.98%), cattle (14.6%), horses (8.43%), small rodents (6.74%), swine (3.93%) and felines (3.37%).", "From October 2014 to April 2016, a total of 648 fecal samples of wild and domestic animals belonging to three forest fragments areas were tested for the NSP3 gene by qualitative qPCR, and 178 (27.5%) were positive for RVA, distributed among the species: birds (23.6%), canines (21.35%), bats (17.98%), cattle (14.6%), horses (8.43%), small rodents (6.74%), swine (3.93%) and felines (3.37%). The CT interval ranged from 28. 47 It was possible to detect viral strains in all genders of animals studied and in the harvesting period none of the animals showed signs of acute infection and / or diarrhea.", "47 It was possible to detect viral strains in all genders of animals studied and in the harvesting period none of the animals showed signs of acute infection and / or diarrhea. Rotavirus A (RVA) detected in the present study of wild and domestic animals belonging to the three areas of forest fragment, according to Fig 2. In relation to the evaluated bovines, only in the city of Viseu, these species were studied because they were created extensively.", "In relation to the evaluated bovines, only in the city of Viseu, these species were studied because they were created extensively. In addition, most of the animals were young with ages varying from 1 day to 8 yearsold, history of deficient vaccination, lack of technical assistance and raised in the form of subsistence. The animals showed no symptoms of diarrhea, only low weight performance and poor sanitary management status.", "The animals showed no symptoms of diarrhea, only low weight performance and poor sanitary management status. In relation to chiroptera, 32 (17.98%) positive samples for RVA were distributed among Carollia perspicillata species, with 12 (37.5%) being all adults, 9 (28.12%) Desmodus rotundus samples (4 young and 5 adults), 5 (15.6%) of Uroderma bilobata (15.62%), 3 (9.37%) of Artibeus lituratus and the species Artibeus Planirostus, Diaemus iyoug and Glossophagine with 1 (3.12%) each. These animals came from areas of forest fragments located near bovine and equine farms, in addition to inhabiting small chicken farms.", "These animals came from areas of forest fragments located near bovine and equine farms, in addition to inhabiting small chicken farms. Fig 3 shows the results obtained for all the species of animals investigated in the forest fragment as well as in the peridomicillus area. The anthropic variables were analyzed for the three cities studied, as well as the use of the soil within the range of the animals, obeying the domicile, the peridomicile and the forest fragment where the traps of small rodents, birds and various species of animals were captured (Fig 4 and Fig 5) .", "The anthropic variables were analyzed for the three cities studied, as well as the use of the soil within the range of the animals, obeying the domicile, the peridomicile and the forest fragment where the traps of small rodents, birds and various species of animals were captured (Fig 4 and Fig 5) . Considering the factors related to the anthropic activities in the three studied areas within the three cities of the present study, it was observed that the city of Santa Bárbara is the one that has a better area of preserved forest and the city of Viseu a smaller area. However, in the city of Santa Bárbara, a greater concentration of occupations was observed around the area of forest fragment.", "However, in the city of Santa Bárbara, a greater concentration of occupations was observed around the area of forest fragment. It was observed in this chosen area of the city, the presence of different families living in a rural settlement, surviving from the exploitation of forest resources and the creation of small animals for subsistence, such as poultry and fish farming, as well as family farming products. The breeding of animals in native pastures was only observed in the cities of Peixe Boi and Viseu.", "The breeding of animals in native pastures was only observed in the cities of Peixe Boi and Viseu. Extensive livestock farming was practiced with beef cattle, equines for work and small animals (swine and goats). In relation to the most preserved pasture area, the city of Peixe Boi had the largest area, according to the data shown in Fig 5, however, in the city of Viseu, a higher regeneration was observed in the pastures during the period of the study, with significant secondary vegetation.", "In relation to the most preserved pasture area, the city of Peixe Boi had the largest area, according to the data shown in Fig 5, however, in the city of Viseu, a higher regeneration was observed in the pastures during the period of the study, with significant secondary vegetation. When comparing the climates of the three areas it was observed that the predominant climate is megathermal and humid with average annual temperature around 27˚C. The months of October, November and December are the hottest, with temperatures between 32˚C and 34˚C and absolute maximums around 41˚C.", "The months of October, November and December are the hottest, with temperatures between 32˚C and 34˚C and absolute maximums around 41˚C. Annual rainfall is quite high, generally around 2,350 mm, but strongly concentrated from January to June (80%). From September to December, on the contrary, rainfall is rare, about 7%, with a short dry season, of moderate water deficit in those months.", "From September to December, on the contrary, rainfall is rare, about 7%, with a short dry season, of moderate water deficit in those months. The relative humidity of the average air oscillates around 85%, as shown in Fig 6 [48] . The description of the accumulated precipitation in the year of capture of the fecal specimens compared to the Climatological Normals (CLINO) for the period from 1961-1990 of the PCDs closest to the locations of the Expedito Ribeiro / Santa Bárbara settlement (Belém PCD), Vila Ananim / Peixe-Boi and Açaiteua / Viseu (Tracauateua PCD) show the frequency of rainfall in the regions, which facilitates the renewal of the pastures and the regeneration of the impacted forests, being an important indicator of the reduction of the damages caused by deforestation in the region.", "The description of the accumulated precipitation in the year of capture of the fecal specimens compared to the Climatological Normals (CLINO) for the period from 1961-1990 of the PCDs closest to the locations of the Expedito Ribeiro / Santa Bárbara settlement (Belém PCD), Vila Ananim / Peixe-Boi and Açaiteua / Viseu (Tracauateua PCD) show the frequency of rainfall in the regions, which facilitates the renewal of the pastures and the regeneration of the impacted forests, being an important indicator of the reduction of the damages caused by deforestation in the region. The average deforestation index in the three study areas was calculated from data obtained from INPE information systems. It was observed that in the years of 2013 to 2014 there were no changes in these regions; in the period from 2014 to 2015 about 4.1% of the city of Viseu was changed and 1.6% of the city of Peixe Boi.", "It was observed that in the years of 2013 to 2014 there were no changes in these regions; in the period from 2014 to 2015 about 4.1% of the city of Viseu was changed and 1.6% of the city of Peixe Boi. In relation to the period of 2016, great changes were observed in Peixe Boi (79%) and in the city of Viseu (70%), thus demonstrating that changes in the natural ecosystem may be associated with the frequencies for RVA in the studied areas, according to Fig 7. When assessing the infected animals in relation to the uninfected animals in both the forest fragment and the peridomicile, considering as animals of the forest fragment the birds, the chiroptera and the small rodents and as animals of the peridomicile the canines, bovines, pigs, felines and horses, a percentage of 37.07% infected peri domestic animals (86/232) and 22.12% infected forest fragment animals (92/416) were obtained.", "When assessing the infected animals in relation to the uninfected animals in both the forest fragment and the peridomicile, considering as animals of the forest fragment the birds, the chiroptera and the small rodents and as animals of the peridomicile the canines, bovines, pigs, felines and horses, a percentage of 37.07% infected peri domestic animals (86/232) and 22.12% infected forest fragment animals (92/416) were obtained. Applying the selected statistical analysis, a Pearson x2 Chi-square value was obtained: 16.7159, df = 1 and p <0.001, meaning that the hypothesis was corroborated, that is, the greater the degradation of the environment, the more likely it will be the search for food by wild animals in adjacent areas, or in the edge of the forest or even in the peri domiciliary region. In this sense, the possibility of contagion with other species of animals, even humans, should be considered because of the capacity of the rotavirus to be transmitted via the fecal / oral route or through direct contact with the environment.", "In this sense, the possibility of contagion with other species of animals, even humans, should be considered because of the capacity of the rotavirus to be transmitted via the fecal / oral route or through direct contact with the environment. It is important to point out that the animals detected in this study are important sources of viral strains. A total of 80 stool samples were selected, reextracted and analyzed using PCR for the VP4 gene.", "A total of 80 stool samples were selected, reextracted and analyzed using PCR for the VP4 gene. Eight strains (10%) were positive for VP4 gene, being 2 strains bellowed to P [6] genotype and 6 to P[4]-type, according to \n\nIn the present study, RVA was detected circulating in 27.5% of the animals; 36% in domestic animals and 64% in wild animals, providing a unique dataset with qRT-PCR detecting a low viral load of RVA in different species, which further correlates with the deforestation index. These data are important because there is a lack of tests for RVA diagnosis in animals, since the current methods of RVA detection does not always detect in these populations [8] .", "These data are important because there is a lack of tests for RVA diagnosis in animals, since the current methods of RVA detection does not always detect in these populations [8] . With the advent of real-time PCR (qPCR), there was an exponential growth, compared to conventional PCR essays, since its superior accuracy, sensitivity and specificity is remarkable, and it is Rotavirus A in wild and domestic animals possible to detect RVA in a variety of animal species using NSP3 gene [49] . The sensitivity of RT-qPCR significantly improved the rate of RVA detection in clinical samples from animals and in this context, the present study proposed an interesting study metrics using virus spreading in the wild animals which inhabit forest fragments to indicate human population interventions, with the goal of preventing the virus outbreaks leveraged on the unique geographic characteristics of Brazil and its large number of species in Amazon.", "The sensitivity of RT-qPCR significantly improved the rate of RVA detection in clinical samples from animals and in this context, the present study proposed an interesting study metrics using virus spreading in the wild animals which inhabit forest fragments to indicate human population interventions, with the goal of preventing the virus outbreaks leveraged on the unique geographic characteristics of Brazil and its large number of species in Amazon. Currently, no data have been described in the literature regarding the RVA detection using real-time qPCR technique in a wide variety of wild animal species. However, a study by Soltan et al.", "However, a study by Soltan et al. [50] conducted with horses and cattle detected RVA by RT-PCR, commercial RT-PCR and RT-qPCR in 36.7%, 51.4% and 56.9% respectively, differently from the present study that showed higher positivity for chiropterans (17.98%), canines (21.35%), birds (23.6%) and cattle (14.6%). The first description of RVA in chiroptera was recorded in feces of Eidolon helvum caught in Vihiga, Kenya [51] .", "The first description of RVA in chiroptera was recorded in feces of Eidolon helvum caught in Vihiga, Kenya [51] . Afterwards, several strains of RVA were detected by different molecular techniques involving chiroptera, in several countries, including Kenya (E. helvum), China (Rhinolophus hipposideros and Aselliscus stoliczkanus), France (Myotis mystacinus), Cameroon (E. helvum) and Brazil [31, [51] [52] [53] [54] [55] . The present study shows the occurrence of RVA in 17.98% of the chiroptera, being among the species Carollia perspicillata (37.5%), Desmodus rotundus (28.12%), Uroderma bilobata (15.6%), Artibeus lituratus (9.37%), Artibeus Planirostus (3.12%), Diaemus iyoug and Glossophagine (3.12%).", "The present study shows the occurrence of RVA in 17.98% of the chiroptera, being among the species Carollia perspicillata (37.5%), Desmodus rotundus (28.12%), Uroderma bilobata (15.6%), Artibeus lituratus (9.37%), Artibeus Planirostus (3.12%), Diaemus iyoug and Glossophagine (3.12%). Barquez et al. [56] reported that Desmodus rotundus is one of the three hematophagous species of the Phyllostomidae family, found throughout South America, Central America and Mexico.", "[56] reported that Desmodus rotundus is one of the three hematophagous species of the Phyllostomidae family, found throughout South America, Central America and Mexico. Of the positive chiroptera for RVA in the present study, a prevalence of 28.12% was of Desmodus rotundus. This species feeds on birds, can feed on mammals, mostly medium or large, facilitating the dissemination of viral spores among the community within the habitat, as observed in the present study.", "This species feeds on birds, can feed on mammals, mostly medium or large, facilitating the dissemination of viral spores among the community within the habitat, as observed in the present study. These findings show the importance of epidemiological data on the studied species due to the lack of studies involving species of neotropical chiroptera, and it is not possible to establish comparative parameters for these animals. Regarding the circulation of RVA in canines and birds, the prevalence was 53% and 29%, respectively.", "Regarding the circulation of RVA in canines and birds, the prevalence was 53% and 29%, respectively. Although in the Amazon region there are records of RVA, RVD, RVF and RVG that infect birds [57] [58] and RVD in migratory birds [59] , all were detected by RT-PCR assays differently from the present study which detected the RVA by RT-qPCR involving a variety of animal species. On the other hand, the prevalence in felines (16%) and pigs (22%) was lower, probably because there are few animals of these species in the region, as well as few creations.", "On the other hand, the prevalence in felines (16%) and pigs (22%) was lower, probably because there are few animals of these species in the region, as well as few creations. The study detected the presence of RVA in different species of animals both in areas near the home and in areas located in fragments of forest, characterized as forest remnants, since they were located in cities that suffered high environmental impacts due to vegetal extractivism, pasture formation for cattle breeding, exploitation of natural resources, and direct reflexes on the habitats of wild animals that can serve as virus sources, thus facilitating the dispersion of RVA among communities of coexisting animals. It is worth emphasizing that these animals have a greater contact with the human populations of the studied areas since they cohabit with the humans in the region, besides having a high flow of movement between the forest extracts and environments chosen for the present study.", "It is worth emphasizing that these animals have a greater contact with the human populations of the studied areas since they cohabit with the humans in the region, besides having a high flow of movement between the forest extracts and environments chosen for the present study. However, it is noteworthy that only in the communities of Santa Bárbara and Viseu were collected fecal specimens from asymptomatic humans for diarrhea and tested for RVA, but all were negative. It is notorious yet, the existence of different levels of degradation in the studied environments, considering the presented data.", "It is notorious yet, the existence of different levels of degradation in the studied environments, considering the presented data. The fragmentation of the forest generates many consequences on the Amazonian biota, being able to alter the diversity and the composition of the animal communities in the fragments and even to interfere in the ecological processes, without considering that the fragments of forest in the Amazon are influenced by the climate, possibly facilitating the dispersion of pathogens by the environment, since the wild animals detected in the present study are asymptomatic and have low viral load for RVA. The occurrence of RVA in this population of animals may explain the possibility of dispersion of viral strains, since there is a proximity to the human population, besides the biological characteristics of these species that may represent important sources for gastroenteric viruses, along with the fact that all animals were asymptomatic for diarrhea.", "The occurrence of RVA in this population of animals may explain the possibility of dispersion of viral strains, since there is a proximity to the human population, besides the biological characteristics of these species that may represent important sources for gastroenteric viruses, along with the fact that all animals were asymptomatic for diarrhea. Wild birds have unlimited flight capacity, were captured in an interface region between the peridomicile and forest fragments and it is believed that this region has not been influenced by anthropic activities such as those observed in the area of the present study. On the other hand, the breeding method for poultry and canines close to homes and the forest ecosystem, as they are created in the communities surveyed, probably facilitates direct contact with possible sources of contamination, since in the areas the use of septic tanks is deficient and sometimes non-existent, which may facilitate or even increase the risk of viral dispersion throughout the environment.", "On the other hand, the breeding method for poultry and canines close to homes and the forest ecosystem, as they are created in the communities surveyed, probably facilitates direct contact with possible sources of contamination, since in the areas the use of septic tanks is deficient and sometimes non-existent, which may facilitate or even increase the risk of viral dispersion throughout the environment. The high rates of increase and the analysis of land use in the researched areas may be important indicators of how these animals interact, since with deforestation, the populations of wild animals seek refuge in nearby communities facilitating the dispersal of infectious agents and the possible occurrence of carrier animals by direct contact or contamination of the local environment. To our knowledge, this is the first study in which a real-time PCR assay was applied for the detection of RVA involving a wide variety of domestic and wild animals, facilitating practical utility in epidemiological and molecular studies and assisting in a perspective in the elaboration of sanitary control and monitoring, preventing possible outbreaks in the studied communities.", "To our knowledge, this is the first study in which a real-time PCR assay was applied for the detection of RVA involving a wide variety of domestic and wild animals, facilitating practical utility in epidemiological and molecular studies and assisting in a perspective in the elaboration of sanitary control and monitoring, preventing possible outbreaks in the studied communities. The detection of positive animals was useful to monitor the infection of the agent in the animal population and to provide an early warning signal to predict an impending epidemic and a favorable risk for the human population, given the evidence of RVA circulation in the different forest fragments. In addition, the RT-qPCR assay may be a useful alternative for the differential diagnosis of RV in possible coexisting mixed infections clinically indistinguishable such as those caused by other viral strains that cause gastroenteritis such as: astrovirus, coronavirus, picobirnavirus, calicivirus, among others as observed in the studies of Jing et al.", "In addition, the RT-qPCR assay may be a useful alternative for the differential diagnosis of RV in possible coexisting mixed infections clinically indistinguishable such as those caused by other viral strains that cause gastroenteritis such as: astrovirus, coronavirus, picobirnavirus, calicivirus, among others as observed in the studies of Jing et al. [60] and Waruhiu et al. [61] .", "[60] and Waruhiu et al. [61] . Diarrhea associated with RVA infections in pigs is an important cause of increased mortality and economic losses in Europe.", "Diarrhea associated with RVA infections in pigs is an important cause of increased mortality and economic losses in Europe. The most prevalent genotypes isolated from feces of Belgian diarrheal and non-diarrheal piglets in 2012 [62] demonstrate a wide range of combinations of genotypes G / P including; G3P [6] , G4P [6] , G5P [6] , G4P [7] , G5P [7] , G9P [7] , G9P [13] and G9P [23] . On the other hand, in the present study it was possible to detect only P [6] genotype, since majority of samples was asymptomatic for diarrhea.", "On the other hand, in the present study it was possible to detect only P [6] genotype, since majority of samples was asymptomatic for diarrhea. Finding shows that different P genotypes of RVA strains interact with distinct blood group histological antigens (HBGA, ABOH, Lewis) and sialic acids via VP4 providing insight into the regional prevalence and increased zoonotic potential of some RVA of origin swine [63] . The genotype P [6] was identified in piglets in Brazil [64] and in Italy and Japan resembling genotype P [6] human [65, 66] .", "The genotype P [6] was identified in piglets in Brazil [64] and in Italy and Japan resembling genotype P [6] human [65, 66] . In the population of animals studied the zoonotic transmission can be frequent, since the animals live in contact with humans and in precarious sanitary conditions. In Brazil, this genotype was described in animal and human populations in studies of Luchs et al.", "In Brazil, this genotype was described in animal and human populations in studies of Luchs et al. [32] ; Honma et al. [67] ; Araújo et al. [68] ; Mascarenhas et al. [69] and Lorenzetti et al.", "[68] ; Mascarenhas et al. [69] and Lorenzetti et al. [70] such studies corroborate the importance of continuing to monitor genotypes to verify if uncommon strains or new strains are emerging and can infect animal populations or inter-species transmissions.", "[70] such studies corroborate the importance of continuing to monitor genotypes to verify if uncommon strains or new strains are emerging and can infect animal populations or inter-species transmissions. Regarding the genotype P [4] , itwas most detected in our samples in bats, dogs, swine and feline. This genotype is not common in animals, being more detected in human and environmental samples in various parts of the world and included our region [71] .", "This genotype is not common in animals, being more detected in human and environmental samples in various parts of the world and included our region [71] . It is important to emphasize that the indicators of environmental contamination in Brazil are significant and contribute to the possibility of human-animal transmission [71] . Such data need further investigation in later work to better characterize the interspecies transmission, since the occurrence of enteric viruses in different matrices demonstrates the anthropogenic impact of the exposed population around and points to the potential risk of infection by the possible exposure of individuals susceptible.", "Such data need further investigation in later work to better characterize the interspecies transmission, since the occurrence of enteric viruses in different matrices demonstrates the anthropogenic impact of the exposed population around and points to the potential risk of infection by the possible exposure of individuals susceptible. Our findings may be useful for tracking fecal contamination in the environment using animals as possible sources thus minimizing the risk of infection by exposure to susceptible individuals, in this case different animal species or even human populations. RVA were detected in wild and domestic animals using a RT-qPCR assay that analyzed samples that had low viral load for RVA.", "RVA were detected in wild and domestic animals using a RT-qPCR assay that analyzed samples that had low viral load for RVA. Although the samples are asymptomatic for diarrhea, it is necessary to conduct strategies for the monitoring and control of the animals in the areas studied in the human population as well as in other species of animals, as well as the implementation of preventive measures aimed at future outbreaks in communities animals in the resident population in these impacted areas. Therefore, the present study is unprecedented in the region and in the country in relation to the research of RVA in wild animals.", "Therefore, the present study is unprecedented in the region and in the country in relation to the research of RVA in wild animals. It is noteworthy that, although the quality of the analyzed samples is characterized as low detectable viral load, the technique presented a good analytical response in the detection of the source animals for RVA, facilitating the selection of the samples for future genetic characterization tests." ]

[ "We investigated the ability of monoclonal B cells to restore primary and secondary T-cell dependent antibody responses in adoptive immune-deficient hosts. Priming induced B cell activation and expansion, AID expression, antibody production and the generation of IgM(+)IgG(-) and IgM(-)IgG(+) antigen-experienced B-cell subsets that persisted in the lymphopenic environment by cell division. Upon secondary transfer and recall the IgM(-)IgG(+) cells responded by the production of antigen-specific IgG while the IgM(+) memory cells secreted mainly IgM and little IgG, but generated new B cells expressing germinal center markers.", "Upon secondary transfer and recall the IgM(-)IgG(+) cells responded by the production of antigen-specific IgG while the IgM(+) memory cells secreted mainly IgM and little IgG, but generated new B cells expressing germinal center markers. The recall responses were more efficient if the antigenic boost was delayed suggesting that a period of adaptation is necessary before the transferred cells are able to respond. Overall these findings indicate that reconstitution of a functional and complete memory pool requires transfer of all different antigen-experienced B cell subsets.", "Overall these findings indicate that reconstitution of a functional and complete memory pool requires transfer of all different antigen-experienced B cell subsets. We also found that the size of the memory B cell pool did not rely on the number of the responding naïve B cells, suggesting autonomous homeostatic controls for naïve and memory B cells. By reconstituting a stable memory B cell pool in immune-deficient hosts using a monoclonal high-affinity B cell population we demonstrate the potential value of B cell adoptive immunotherapy.", "By reconstituting a stable memory B cell pool in immune-deficient hosts using a monoclonal high-affinity B cell population we demonstrate the potential value of B cell adoptive immunotherapy. Text: Immune responses to infectious agents have different out-comes that can either protect or fail to control disease. Protection from re-infection relies on the establishment of efficient secondary immune responses that require the generation of antigen-specific \"memory\" B and T lymphocytes.", "Protection from re-infection relies on the establishment of efficient secondary immune responses that require the generation of antigen-specific \"memory\" B and T lymphocytes. The generation and selection of T-cell dependent \"memory\" B cells involves distinct molecular mechanisms: immunoglobulin isotype recombination and somatic hyper mutation, both dependent on the expression of AID [1] . Therefore, a long-standing paradigm defined memory B cells as IgM -IgG + isotype switched cells [2] .", "Therefore, a long-standing paradigm defined memory B cells as IgM -IgG + isotype switched cells [2] . Different lines of evidence indicate that this is not always the case. In humans, it has been shown that some IgM + B cells bear the phenotype of other memory cells, being CD27 + , and carry frequent point mutations in the V region of the Ig genes, suggesting that they must represent highly selected B cell populations [3] .", "In humans, it has been shown that some IgM + B cells bear the phenotype of other memory cells, being CD27 + , and carry frequent point mutations in the V region of the Ig genes, suggesting that they must represent highly selected B cell populations [3] . In mice, populations of CD19 + IgM + able to mount secondary responses have been identified [4] [5] [6] [7] . Overall these findings suggest that the T-cell dependent memory B cell pool comprises distinct subsets of memory B cells with different properties and effector functions [4] [5] [6] .", "Overall these findings suggest that the T-cell dependent memory B cell pool comprises distinct subsets of memory B cells with different properties and effector functions [4] [5] [6] . The biological properties that ensure the long-term persistence of memory and efficient secondary antibody responses have not been yet completely established. While initial studies proposed that after transfer memory B cells faded rapidly [8, 9] suggesting that long-lasting memory required the continuous recruitment of new cells [8] and/or antigen persistence [9, 10] , others suggested that memory B cells were able of extended survival without cell division [11] in the absence of antigen [2] .", "While initial studies proposed that after transfer memory B cells faded rapidly [8, 9] suggesting that long-lasting memory required the continuous recruitment of new cells [8] and/or antigen persistence [9, 10] , others suggested that memory B cells were able of extended survival without cell division [11] in the absence of antigen [2] . Long-term persistence of antibody responses has also been attributed to populations of long-lived plasma cells mainly resident in the bone marrow following immunization [12, 13] . The demonstration of the compartmentalization of \"antibody memory\" into different cellular layers suggested that the separate subsets of memory B cells behave differently.", "The demonstration of the compartmentalization of \"antibody memory\" into different cellular layers suggested that the separate subsets of memory B cells behave differently. Accordingly, it has been reported that IgG + cells that could rapidly respond upon challenge did not persist long, while IgM + cells could generate a second wave of germinal center responses allowing persistence of memory [4] [5] [6] 14] . Currently, immunotherapy approaches using passive antibody transfer [15, 16] ) is limited by the short half-life of immunoglobulin.", "Currently, immunotherapy approaches using passive antibody transfer [15, 16] ) is limited by the short half-life of immunoglobulin. Therefore new therapy strategies may require the adoptive transfer of high-affinity memory B cells, ready to respond and able to persist. The development of these new strategies requires a profound understanding of the mechanisms that regulate memory B cell numbers and ensure long persistence upon adoptive transfer.", "The development of these new strategies requires a profound understanding of the mechanisms that regulate memory B cell numbers and ensure long persistence upon adoptive transfer. Moreover, knowledge of the mechanisms that determine the size of the memory B cell pool may be also critical to device new reconstitution strategies. So far, studies comparing populations of naïve and memory B cells have been hindered both by the vast clonal heterogeneity of the cells involved and by our inability to generate significant numbers of antigen specific memory B cells.", "So far, studies comparing populations of naïve and memory B cells have been hindered both by the vast clonal heterogeneity of the cells involved and by our inability to generate significant numbers of antigen specific memory B cells. Indeed in a normal laboratory mouse the population of B cells bearing a \"memory IgG + phenotype' represent a small fraction of the total B cell pool (<0.5%) and upon immunization the number of the clonal diverse antigen-specific memory B cells generated is generally very limited (<10 3 ) [1, 6] . To circumvent these limits, we decided to compare the properties of homogeneous populations of naïve and memory B cells of known antigen specificity, belonging to the same clone.", "To circumvent these limits, we decided to compare the properties of homogeneous populations of naïve and memory B cells of known antigen specificity, belonging to the same clone. We used SW HEL transgenic mice where B cells bear a high-affinity BCR specific for HEL and are capable of class switch recombination and somatic hypermutation (SHM) [17, 18] . To identify \"memory B cells\" the SW HEL mice were crossed with mice where AID transcription provokes the permanent expression of an YFP reporter in post-germinal center lymphocytes [19] .", "To identify \"memory B cells\" the SW HEL mice were crossed with mice where AID transcription provokes the permanent expression of an YFP reporter in post-germinal center lymphocytes [19] . These mice were in a Rag2-deficient background and therefore contain a pure population of monoclonal HEL-specific B cells. To generate memory cells, purified naïve B cells from the SW HEL .AID/YFP.Rag2 -/mice were transferred into adoptive hosts together with monoclonal OVA-specific CD4 + T cells from OTII.Rag2 -/-TCR transgenic mice.", "To generate memory cells, purified naïve B cells from the SW HEL .AID/YFP.Rag2 -/mice were transferred into adoptive hosts together with monoclonal OVA-specific CD4 + T cells from OTII.Rag2 -/-TCR transgenic mice. Upon immunization with OVA-HEL complexes, we obtained a significant number of persisting HEL-specific IgM + Ig-G -YFP + and IgM -IgG + YFP + memory B cells, number that did not correlate to the number of precursor naïve cells initially injected suggesting that the memory B cell pool is regulated independently. We characterized the functional capacity of these two memory cell types in immune deficient hosts.", "We characterized the functional capacity of these two memory cell types in immune deficient hosts. Mice B6 and B6.Rag2-/- [20] mice were kept at the Centre Des Techniques Avancées (CDTA), Centre National de la Recherche Scientifique (CNRS), Orleans, France; SWHEL.AID/YFP.Rag-/-mice, obtained by crossing SWHEL (18)(a gift of Dr. Robert Brink) and AID/YFP [19] (a gift of Dr. Rafael Casellas) with B6.Rag2-/-mice. OTII.Rag-/-mice were kept in our animal facilities at the Pasteur Institute.", "OTII.Rag-/-mice were kept in our animal facilities at the Pasteur Institute. Experiments were preformed according to Pasteur Institute Safety Committee in accordance with French and European guidelines and the ethics Committee of Paris 1 (permits 2010-0002, -0003 and -0004). Euthanasia of the mice was performed by cervical dislocation.", "Euthanasia of the mice was performed by cervical dislocation. This specific study was approved by the European Research Council (ERC) committee related to the grant AdG09 249740-QSIS. The general status of the mice was controlled daily by monitoring the appearence of obvious pain, distress or suffering (prostration, respiratory issues, loss of weight).", "The general status of the mice was controlled daily by monitoring the appearence of obvious pain, distress or suffering (prostration, respiratory issues, loss of weight). The end-point of the experiment was determined by a loss of more than 20% of the weight or as soon as the distress signs appeared. In this case, experiment was stopped and the animals were euthanized.", "In this case, experiment was stopped and the animals were euthanized. Single-cell suspensions of B cells from spleens and lymph nodes of SW HEL .AID/YFP.Rag -/mice together with CD4 + T cells from spleens and lymph nodes of OTII.Rag -/mice were transferred intravenously into the retro-orbital sinus of B6.Ly5 a IgH a or B6.Rag2 -/recipient mice. Mice received 10 6 HEL + B cells and 10 6 CD4 + T cells unless stated otherwise.", "Mice received 10 6 HEL + B cells and 10 6 CD4 + T cells unless stated otherwise. Mice were immunized 24H later with 1 mg of Ovalbumin coupled to Hen Egg Lysozyme (OVA-HEL) in 50μg of Alu-S-Gel (Serva) we determined as the optimal dose of Ag (data not shown). Naive cells from SW HEL .AID/YFP.Rag -/mice and memory B cells subsets from immunized B6.Rag -/hosts mice were purified from spleens and lymph nodes by flow cytometry sorting.", "Naive cells from SW HEL .AID/YFP.Rag -/mice and memory B cells subsets from immunized B6.Rag -/hosts mice were purified from spleens and lymph nodes by flow cytometry sorting. Single-cell suspensions containing 5×10 4 B cells and 10 6 T cells were transferred intravenously into B6.Rag2 -/recipient hosts. The purity of sorted cells was above 98%.", "The purity of sorted cells was above 98%. 24 h after transfer, mice were immunized with 1 mg of OVA-HEL. Spleen, bone marrow, inguinal and mesenteric lymph nodes single-cell suspensions were stained for cell surface or intracellular proteins with appropriate combinations of the following monoclonal antibodies conjugated to pacific blue, Qdot-655, Brillant Violet 605, allophycocyanin, peridinin chlorophyll protein-cyanine 5.5, phycoerythrin, phycoerythrin-cyanine7: anti-CD19 (6D5), anti-IgM (R6-60.2), anti-IgG1 (X56), anti-CD138 (281-2), anti-Gl7 (Gl7), anti-CD95 (Jo2), anti-CD62L (MEL-14), anti-CD69 (H1-2F3), anti-BAFFR (7H22-E16), anti-CXCR5 (L138D7), anti-IA b (AF6-120.1), anti-CD80 (16-10A1), anti-CD73 (TY-11-8) and anti-PDL2 (TY25) and anti-Ki-67 (mm1) purchased from Becton Dickinson Pharmingen, Biolegend, Invitrogen and eBioscience.", "Spleen, bone marrow, inguinal and mesenteric lymph nodes single-cell suspensions were stained for cell surface or intracellular proteins with appropriate combinations of the following monoclonal antibodies conjugated to pacific blue, Qdot-655, Brillant Violet 605, allophycocyanin, peridinin chlorophyll protein-cyanine 5.5, phycoerythrin, phycoerythrin-cyanine7: anti-CD19 (6D5), anti-IgM (R6-60.2), anti-IgG1 (X56), anti-CD138 (281-2), anti-Gl7 (Gl7), anti-CD95 (Jo2), anti-CD62L (MEL-14), anti-CD69 (H1-2F3), anti-BAFFR (7H22-E16), anti-CXCR5 (L138D7), anti-IA b (AF6-120.1), anti-CD80 (16-10A1), anti-CD73 (TY-11-8) and anti-PDL2 (TY25) and anti-Ki-67 (mm1) purchased from Becton Dickinson Pharmingen, Biolegend, Invitrogen and eBioscience. Cells were also stained with HEL (Sigma) coupled with AF594 using Alexa Fluor1 594 Protein Labeling Kit from Life technologies. Before staining, cells were treated with Fc-Block (CD16/CD32, Becton Dickinson Pharmingen).", "Before staining, cells were treated with Fc-Block (CD16/CD32, Becton Dickinson Pharmingen). Dead cells were excluded during analysis according to their light-scattering characteristics. For intracellular stainings, cells were first stained with antibodies specific for cell surface antigens. Then, cells were fixed and permeabilized according the manufacturer's recommendations (BD Bisciences).", "Then, cells were fixed and permeabilized according the manufacturer's recommendations (BD Bisciences). For proliferation assay, mice were injected i.p. with 50 mg/kg of BrdU (Sigma-Aldrich) and were killed 24 or 72 hours later. Incorporated BrdU was detected intracellularly using anti-BrdU APC-conjugated antibodies according to the manufacturer's recommendations (BD Biosciences).", "Incorporated BrdU was detected intracellularly using anti-BrdU APC-conjugated antibodies according to the manufacturer's recommendations (BD Biosciences). All data acquisitions and analyses were performed with LSRFortessa (Becton Dickinson) interfaced with BD FACSDiva (Becton Dickinson) and FlowJo (Tree Star) software. Subsets of memory B cells were sorted as CD19 + HEL + YFP + IgM + or IgG + and naive cells as CD19 + HEL + YFP -IgM + using a FACSAriaIII flow cytometer.", "Subsets of memory B cells were sorted as CD19 + HEL + YFP + IgM + or IgG + and naive cells as CD19 + HEL + YFP -IgM + using a FACSAriaIII flow cytometer. The purity of the sorted populations varied from 90-95%. Sera HEL-specific Ig concentrations were quantified by ELISA.", "Sera HEL-specific Ig concentrations were quantified by ELISA. Plates were coated with HEL and saturated with PBS-5% Milk. Dilutions of sera were added. After incubation (2 hours, 37˚C) and washing, HRP-labeled anti-mouse IgM or IgG antibodies were added. After incubation and washing, bound antibodies were revealed with the substrate O-phenylenediamine and H2O2.", "After incubation and washing, bound antibodies were revealed with the substrate O-phenylenediamine and H2O2. The reaction was stopped after 10 min. by addition of 10% SDS and the absorbance read at 492nm in a multiscan spectrometer.", "by addition of 10% SDS and the absorbance read at 492nm in a multiscan spectrometer. Ig concentrations were determined by comparing the displacement of the dilution curves in the linear interval between standards at a concentration of 1 mg/ml and the serum samples. The quantification of IgG or IgM secreting cells was assayed by ELISpot technique.", "The quantification of IgG or IgM secreting cells was assayed by ELISpot technique. Briefly, plates were coated with HEL. After saturating, the cells were distributed into the micro wells in RPMI1640-2%FCS. The plates were incubated for 12 h at 37˚C, 5% CO2 atmosphere.", "The plates were incubated for 12 h at 37˚C, 5% CO2 atmosphere. After extensive wash, plates were incubated with goat anti-mouse IgM or anti-IgG labeled with alkaline phosphatase. After washing, the revealing substrate was added (2,3 mM 5-bromo-4-chloro-3-indolyl phosphate diluted in 2-amino-2-methyl-1-proprenolol buffer).", "After washing, the revealing substrate was added (2,3 mM 5-bromo-4-chloro-3-indolyl phosphate diluted in 2-amino-2-methyl-1-proprenolol buffer). Spleens from 14 day-immunized mice were initially fixed with paraformaldehyde and embedded in 4% low-gelling-temperature agarose (type VII-A; Sigma-Aldrich) prepared in PBS. 150μm slices were cut with a vibratome (VT 1000S; Leica) in a bath of ice-cold PBS.", "150μm slices were cut with a vibratome (VT 1000S; Leica) in a bath of ice-cold PBS. For immunolabeling, samples were saturated with PBS supplemented with 10% of fetal calf serum, then were labeled with primary antibodies anti-B220-APC (clone RA3-6B2) and anti-IgD-PE (clone 11-26c.2a) and analyzed with a spinning disk confocal microscope equipped with a CoolSnap HQ2 camera (Photometrics) and a 20x objective. Images were acquired and analyzed with MetaMorph 7 imaging software Molecular Devices).", "Images were acquired and analyzed with MetaMorph 7 imaging software Molecular Devices). Sample means were compared using the Student's t test. Sample means were considered significantly different at p < 0.05.", "Sample means were considered significantly different at p < 0.05. During an immune response the complexity of determinants expressed by immunizing antigen and the degeneracy of antigen-specific recognition results in a vast heterogeneity of responding cells rendering impossible the direct comparison of the properties of naïve and memory B cells belonging to the same clone. We have devised an experimental system that permits the comparison between naïve and memory B cells expressing the same antigen receptor and allows marking permanently memory B cells.", "We have devised an experimental system that permits the comparison between naïve and memory B cells expressing the same antigen receptor and allows marking permanently memory B cells. For that purpose we used SW HEL transgenic mice in a Rag2-deficient background holding a single population of monoclonal B cells, all bearing a high-affinity BCR specific for HEL and capable of class switch recombination and somatic hypermutation (SHM) [17, 18] . To identify antigen-experienced B cells the SW HEL .", "To identify antigen-experienced B cells the SW HEL . Rag2 -/mice were crossed with mice where AID transcription induces the permanent expression of an YFP reporter in post-germinal center lymphocytes [19] . Since in intact Tg mice immune responses were not traceable, probably because of the presence of low level pre-existing anti-HEL antibodies that neutralize the immunizing protein, we used an adoptive cell transfer strategy to study the ability of the high affinity monoclonal B cell to reconstitute response in immune-deficient hosts and generate antibody memory.", "Since in intact Tg mice immune responses were not traceable, probably because of the presence of low level pre-existing anti-HEL antibodies that neutralize the immunizing protein, we used an adoptive cell transfer strategy to study the ability of the high affinity monoclonal B cell to reconstitute response in immune-deficient hosts and generate antibody memory. Purified naïve B cells from the SW HEL .AID/YFP.Rag2 -/mice were transferred into Rag2-deficient mice together with monoclonal OVA-specific CD4 + T helper cells from OTII.Rag2 -/-TCR transgenic mice. The day after, host mice were immunized with OVA-HEL complexes (Fig 1A) .", "The day after, host mice were immunized with OVA-HEL complexes (Fig 1A) . In these conditions, antigenic challenge resulted in B cell activation and the development of significant numbers of CD19 + HEL + AID/YFP + B cells, which were not detected in non-immunized mice or in mice immunized in absence of helper T cells (Fig 1B) . We followed the early kinetics of this response.", "We followed the early kinetics of this response. The number of HEL-specific B cells increased from the initial 2x10 6 transferred to about 15x10 6 at day 14 (Fig 1C left) the B cells expressing AID/YFP being the dominant population (Fig 1C right) . A fraction of the HEL-specific B cells underwent class switch recombination and at day 14 we recovered both IgM + IgG -AID/YFP + and IgM -IgG + AID/YFP + cell populations (Fig 1B) .", "A fraction of the HEL-specific B cells underwent class switch recombination and at day 14 we recovered both IgM + IgG -AID/YFP + and IgM -IgG + AID/YFP + cell populations (Fig 1B) . B cell expansion and phenotypic changes were accompanied by the production of IgM and IgG HEL-specific antibodies ( Fig 1D) . Two weeks after antigenic challenge we observed the formation of germinal centers in the spleen of the host mice ( Fig 1E) .", "Two weeks after antigenic challenge we observed the formation of germinal centers in the spleen of the host mice ( Fig 1E) . Coherently we found that while upon adoptive transfer all B cells expressed CD95, only after antigenic challenge most YFP + B cells expressed the germinal center specific marker GL7 (Fig 1F) . In conclusion, the adoptive cell transfer strategy allowed the development of a primary immune response with B cell activation and expansion, induction of AID expression, class switch recombination, antigen-specific IgM and IgG antibody production and germinal center formation.", "In conclusion, the adoptive cell transfer strategy allowed the development of a primary immune response with B cell activation and expansion, induction of AID expression, class switch recombination, antigen-specific IgM and IgG antibody production and germinal center formation. We studied the evolution of the B cell response. From two weeks onwards the total number of B cells contracted and at four weeks we recovered about 2-4x10 6 cells, number that remained stable up to week 20 (Fig 2A) .", "From two weeks onwards the total number of B cells contracted and at four weeks we recovered about 2-4x10 6 cells, number that remained stable up to week 20 (Fig 2A) . High titers of HEL-specific IgG were kept from week 3 to 8, declined thereafter, but were still significantly elevated 20 weeks later (Fig 2B) . A population of cells secreting HEL-specific Igs was present in the spleen (Fig 2C) , but not in the BM (not shown) even at the late time points.", "A population of cells secreting HEL-specific Igs was present in the spleen (Fig 2C) , but not in the BM (not shown) even at the late time points. About 60% of the recovered cells exhibited the phenotype of antigen-experienced (\"memory\") CD19 + HEL + AID/YFP + expressing either IgM or IgG ( Fig 2D and 2E) . We compared the phenotype of the two AID/YFP + IgM + and AID/YFP + IgM -IgG + memory cell populations recovered with that of the naïve B cells (Fig 2F) .", "We compared the phenotype of the two AID/YFP + IgM + and AID/YFP + IgM -IgG + memory cell populations recovered with that of the naïve B cells (Fig 2F) . We found that antigen-experience and naïve B cells expressed similar levels of CD62L, CD69 and BAFFR (not shown). Antigen-experienced cells presented sustained expression of CD95 and increased levels of PNA, but the vast majority lost expression of the germinal center marker GL7 present at earlier times post-immunization ( Fig 2F compare to Fig 1F) .", "Antigen-experienced cells presented sustained expression of CD95 and increased levels of PNA, but the vast majority lost expression of the germinal center marker GL7 present at earlier times post-immunization ( Fig 2F compare to Fig 1F) . Compared to naïve B cells, AID/ YFP + cells expressed higher levels of CD80 and MHC class II and down-regulated expression of CXCR5 (Fig 2F) . These findings indicate that the post-germinal center AID/YFP + B cells express an activated phenotype [5, 21] , have increased antigen-presenting capacity [22] , but may loose the ability to re-enter primary follicles being CXCR5 low [23] .", "These findings indicate that the post-germinal center AID/YFP + B cells express an activated phenotype [5, 21] , have increased antigen-presenting capacity [22] , but may loose the ability to re-enter primary follicles being CXCR5 low [23] . We have also compared the patterns of gene expression (RNAseq) by naïve, activated (YFPcells of immunized mice) and both populations of YFP + memory cells. The data shows a clear discrimination of naïve and activated/memory cells while indicating only minor differences between both subsets of YFP + memory cells (Fig 3) .", "The data shows a clear discrimination of naïve and activated/memory cells while indicating only minor differences between both subsets of YFP + memory cells (Fig 3) . mRNA was isolated from sort-purified Naïve (CD19 + HEL + YFP -IgM + ) IgM + IgGor IgM -IgG + HEL + CD19 + YFP + memory B cells from spleen of different recipient mice. Total recommended by the manufacturer.", "Total recommended by the manufacturer. The validated libraries were then subjected to DNA sequencing. The analysis is performed using the R software, Bioconductor packages including DESeq2 and the PF2tools package (version 1.2.9) developed at PF2 (Institut Pasteur).", "The analysis is performed using the R software, Bioconductor packages including DESeq2 and the PF2tools package (version 1.2.9) developed at PF2 (Institut Pasteur). Normalization and differential analysis are carried out according to the DESeq2 model and package (version 1.8.1). Fig 3A shows a representative heat map of the different cells populations.", "Fig 3A shows a representative heat map of the different cells populations. Fig 3B shows \n\nLate in the immune response persistent B cell numbers were kept by active cell division as a significant fraction of the cells were Ki67 + (Fig 2G left) and incorporated BrdU (Fig 2G middle) . The frequency of BrdU + cells was higher among the AID/YFPcells (15%) than in the major AID/YFP + memory population (3%) and similar between the IgM + and IgM -AID/ YFP + populations ( Fig 2G middle and not shown) .", "The frequency of BrdU + cells was higher among the AID/YFPcells (15%) than in the major AID/YFP + memory population (3%) and similar between the IgM + and IgM -AID/ YFP + populations ( Fig 2G middle and not shown) . Three days after BrdU pulse populations were clear of BrdU + cells (Fig 2G right) attesting their high division rate. In spite of their increased proliferation rate, memory cells numbers were stable indicating that proliferation may be compensated by cell death as suggested by the frequency of caspase3 + cells (Fig 2H) .", "In spite of their increased proliferation rate, memory cells numbers were stable indicating that proliferation may be compensated by cell death as suggested by the frequency of caspase3 + cells (Fig 2H) . The frequency of Caspase3 + cells was higher among the AID/YFP + cells suggesting that a fraction of these cells may represent cells undergoing terminal differentiation. Importantly, these findings demonstrate that the transfer strategy allowed the generation of significant numbers of persisting antigen-experienced YFP + cells.", "Importantly, these findings demonstrate that the transfer strategy allowed the generation of significant numbers of persisting antigen-experienced YFP + cells. It is not yet known whether the number of antigen-experienced memory B cells correlated to the number of naïve B cells or if it is controlled independently of the initial number of antigenspecific B cells present. To approach this question we transferred different numbers of mature naïve B cells from SW HEL .AID/YFP.Rag2 -/donors (ranging from 10 5 to 5.10 6 ) into Rag2-deficient mice together with an excess of CD4 + T helper cells (10 6 ) and immunize the hosts the day after cell transfer with OVA-HEL in optimal non-limiting quantities.", "To approach this question we transferred different numbers of mature naïve B cells from SW HEL .AID/YFP.Rag2 -/donors (ranging from 10 5 to 5.10 6 ) into Rag2-deficient mice together with an excess of CD4 + T helper cells (10 6 ) and immunize the hosts the day after cell transfer with OVA-HEL in optimal non-limiting quantities. To directly compare the results obtained after the transfer of different all numbers we allowed the responses to reach steady-state eight weeks after antigenic challenge. We studied the amplitude of the immune response by measuring the serum titers of HEL-specific IgG antibodies and enumerating the number of HEL-specific B cells recovered.", "We studied the amplitude of the immune response by measuring the serum titers of HEL-specific IgG antibodies and enumerating the number of HEL-specific B cells recovered. We found that in the presence of excess T cell help, the levels of the HEL-specific IgGs (Fig 4C) , and both the total number of HEL-specific ( Fig 4A) and of memory YFP + B cells recovered (Fig 4B) , did not correlate to the number of antigen specific naïve B cells initially transferred. Memory B cells are defined functionally by their ability to induce secondary IgG antibody responses upon secondary antigenic challenge.", "Memory B cells are defined functionally by their ability to induce secondary IgG antibody responses upon secondary antigenic challenge. We investigated whether the subsets of AID/ YFP + IgM + and AID/YFP + IgM -IgG + antigen-experienced (memory) B cells persisting at late time points could mount secondary IgG responses and persist after secondary transfer. For this purpose we followed two different experimental strategies.", "For this purpose we followed two different experimental strategies. In the first, 5x10 4 cells of either IgM + or IgM -IgG + memory B cells, were transferred with an excess helper OTII CD4 + T cells into secondary Rag-deficient hosts that were boosted with OVA-HEL the day after cell transfer. In the absence of immunization antibody levels were undetectable (not shown) and three weeks after transfer recovery of both memory B cell subsets was about 10-20% of the initial cell input, exceeding naïve B cell recovery (Fig 5A) , supporting the notion that memory B cells may not require specific ligand recognition to survive (2).", "In the absence of immunization antibody levels were undetectable (not shown) and three weeks after transfer recovery of both memory B cell subsets was about 10-20% of the initial cell input, exceeding naïve B cell recovery (Fig 5A) , supporting the notion that memory B cells may not require specific ligand recognition to survive (2). One cannot exclude, however, that cross-reactivity of the BCR transgene with environmental antigens may allow signaling sufficient to maintain naïve and memory cell survival in the absence of HEL [24] . Following immunization, the secondarily transferred AID/YFP + IgM -IgG + cells responded promptly with the exclusive production of significant levels HEL-specific IgG thus confirming their memory statute (11) .", "Following immunization, the secondarily transferred AID/YFP + IgM -IgG + cells responded promptly with the exclusive production of significant levels HEL-specific IgG thus confirming their memory statute (11) . The AID/YFP + IgM + B cells in response to antigenic boost produced only limited amounts of IgM antibodies (Fig 4B) , little IgG antibodies, but did generate GL7 + B cells more efficiently than the IgG + memory B cell population (Fig 5D) . Thus the IgM + subset may contain precursors able to generate a secondary germinal center reaction and a new progeny of IgG + effectors (4).", "Thus the IgM + subset may contain precursors able to generate a secondary germinal center reaction and a new progeny of IgG + effectors (4). With time antibody levels decayed rapidly suggesting that the number of transferred memory B cells declined in the secondary hosts after antigenic boost. Indeed, IgM + and IgG + memory B cells failed to expand and 3 weeks after immunization cell recovery was similar to the retrieval observed in the non-immunized hosts (compare Fig 5E and 5A) .", "Indeed, IgM + and IgG + memory B cells failed to expand and 3 weeks after immunization cell recovery was similar to the retrieval observed in the non-immunized hosts (compare Fig 5E and 5A) . In similar experimental conditions, naïve B cells following immunization expanded, acquired AID/ YFP expression and their numbers more than doubled the number initially injected (Figs 5F and 2A). These data suggest that a significant fraction of the memory B cells generated have a reduced expansion capacity being programmed for rapid differentiation for effector functions.", "These data suggest that a significant fraction of the memory B cells generated have a reduced expansion capacity being programmed for rapid differentiation for effector functions. Besides long-term survival memory B cells must maintain functional activity in the absence of nominal antigen to be fully effective. To test this we used an alternative approach where memory cells were parked in secondary Rag-deficient hosts for 30 days before re-immunization.", "To test this we used an alternative approach where memory cells were parked in secondary Rag-deficient hosts for 30 days before re-immunization. We found that under these conditions antigenic challenge resulted in the production of HEL-specific IgG antibodies and in a 100 fold increase in the number of cells recovered, expansion that largely exceed that observed after immediate challenge (Fig 5G) . The aim of this study was to characterize the fate of activated B cells and the generation of memory B cells.", "The aim of this study was to characterize the fate of activated B cells and the generation of memory B cells. To do this, we adoptively transferred monoclonal B cells into immune deficient hosts followed by immunization in presence of T cell help. This strategy resulted in the development of different B cell memory subsets, namely IgM + and IgG + , as described for in situ generated memory cells [4, 6, 14] .", "This strategy resulted in the development of different B cell memory subsets, namely IgM + and IgG + , as described for in situ generated memory cells [4, 6, 14] . These findings indicate that distinct memory B cell subsets are not the result of the heterogeneity of initially responding naive cells, but originate from the differentiation of a single B cell clone. While studying the respective rate of proliferation of both types of memory B cells, we found the same high rate of proliferation for IgM + and IgG + memory B cells.", "While studying the respective rate of proliferation of both types of memory B cells, we found the same high rate of proliferation for IgM + and IgG + memory B cells. These results contrast with previous published data. First it was been reported that \"in situ\" memory B cells persist as resting non-dividing cells [11, 25] .", "First it was been reported that \"in situ\" memory B cells persist as resting non-dividing cells [11, 25] . However, we have shown that upon adoptive transfer and in absence of competing cells, B cells increase their division rate to occupy the available empty niche [26] , which may explain the higher division rate observed here using this adoptive cell transfer strategy. Secondly, comparing life spans among heterogeneous memory B cell populations it was previously reported a lower division rate among the IgM + subset compared to the IgG + polyclonal subset [6] .", "Secondly, comparing life spans among heterogeneous memory B cell populations it was previously reported a lower division rate among the IgM + subset compared to the IgG + polyclonal subset [6] . Differences in BCR affinity between IgM + and IgG + memory clones may explain the higher division rate previously observed among the IgG + cells [6] . In contrast we compared memory B cell subsets belonging to the same clone bearing the same high affinity BCR.", "In contrast we compared memory B cell subsets belonging to the same clone bearing the same high affinity BCR. Overall these observations support the notion that lymphocyte division rates and life spans are not an intrinsic cell property, but rather determined by the environment and the presence of competing populations [27] . They demonstrate that upon the correct conditions memory B cells can persist by cell division.", "They demonstrate that upon the correct conditions memory B cells can persist by cell division. An important question was whether the number of memory B cells depends on the number of initial naïve B cells. We found that, in the presence of an excess of T cell help, that was not the case.", "We found that, in the presence of an excess of T cell help, that was not the case. However, it was previously reported during polyclonal responses that serum titers of anti-HSA was proportional to the number of cells transferred into irradiated mice [28] . It is possible that limited antigen-specific T-B cell encounters may constraint the number of responding B cells and thus determine linear precursor-progeny between naïve and memory B cells.", "It is possible that limited antigen-specific T-B cell encounters may constraint the number of responding B cells and thus determine linear precursor-progeny between naïve and memory B cells. Our findings indicate that within a single clone the number of precursor naive B cells present in the peripheral B cell pool does determine neither the intensity nor the final number of memory B cells in response to an optimal dose of antigen. They suggest that the size of memory B cell pool may be controlled independently of the number of naïve B cell precursors and that in the absence of clonal competition the memory niche can be filled with a single monoclonal population.", "They suggest that the size of memory B cell pool may be controlled independently of the number of naïve B cell precursors and that in the absence of clonal competition the memory niche can be filled with a single monoclonal population. Considering diverse polyclonal populations, the limited niche for memory cells will imply strong competition among clones resulting in the selection of best fit (high affinity) cells: rare mutated clones being able to out compete more frequent but less avid clones. In our settings, the transgenic memory B cells are likely to counter select any new mutant clones since they express a very high affinity BCR selected in the course of a secondary immune response [29] .", "In our settings, the transgenic memory B cells are likely to counter select any new mutant clones since they express a very high affinity BCR selected in the course of a secondary immune response [29] . Thus, notwithstanding the expression of AID and proliferation we did not detect any BCR VH and VL Ig-chain nucleotide mutations among the recovered memory B cells (not shown). These findings may have implication for vaccination protocols as they indicate that each new antigenic exposure or unrelated immunization would add extra competing clones supporting the need for repeated antigenic boosts to prevent memory B cell attrition.", "These findings may have implication for vaccination protocols as they indicate that each new antigenic exposure or unrelated immunization would add extra competing clones supporting the need for repeated antigenic boosts to prevent memory B cell attrition. They also demonstrate that the memory B cell pool can be reconstituted from a relatively small number of antigen-specific cells. It is likely that the relatively poor memory B cell expansion observed after immediate boost after adoptive transfer could be due to the lack in Rag-deficient hosts of the appropriate environment required for memory B cell survival and function.", "It is likely that the relatively poor memory B cell expansion observed after immediate boost after adoptive transfer could be due to the lack in Rag-deficient hosts of the appropriate environment required for memory B cell survival and function. It should be pointed out that B cell transfer into transgenic ML5 Rag-deficient hosts expressing low levels of HEL [29] resulted in rapid cell loss and recovery suggesting that in these hosts, B cells are trapped by antigen in locations were they are unable to survive (not shown). Nevertheless, it has been shown that B cells can drive the maturation of follicular dendritic cells and the organization of lymphoid follicles [30] .", "Nevertheless, it has been shown that B cells can drive the maturation of follicular dendritic cells and the organization of lymphoid follicles [30] . Similarly, transferred helper cells may also modify their immediate environment. Thus, by allowing lymphocytes to adapt and modify their immediate environment we improved their response and more important, we recovered the memory B cell pool size present in the original donor mice.", "Thus, by allowing lymphocytes to adapt and modify their immediate environment we improved their response and more important, we recovered the memory B cell pool size present in the original donor mice. In this study we show that it is possible to fully reconstitute a primary response and the establishment of antibody memory in immune deficient mice after adoptive transfer of antigen-specific monoclonal B cells together with a population of monoclonal helper T cells. Indeed, it is generally believed that in immune deficiencies, B cell therapy has restricted application due to intrinsic defects of host's lymphoid organs structure that may prevent development of immune responses, germinal center formation, establishment of antibody memory and limit cell survival.", "Indeed, it is generally believed that in immune deficiencies, B cell therapy has restricted application due to intrinsic defects of host's lymphoid organs structure that may prevent development of immune responses, germinal center formation, establishment of antibody memory and limit cell survival. In contrast we showed that after adoptive transfer in immune deficient hosts antigen immunization induced B cell activation and expansion, induction of AID expression, class switch recombination, antigen-specific IgM and IgG antibody production, germinal center formation and the generation of two subsets of AID/YFP + IgM + IgGand AID/YFP + Ig-M -IgG + antigen-experienced B cell subsets able to persist in a lymphopenic environment by cell division mimicking responses obtained in intact non-Tg mice [4] . Upon challenge the AID/YFP + IgM -IgG + cells responded promptly with the production of HEL-specific IgG while the AID/YFP + IgM + B cells secreted only limited amounts of IgM antibodies and fail to produce IgG.", "Upon challenge the AID/YFP + IgM -IgG + cells responded promptly with the production of HEL-specific IgG while the AID/YFP + IgM + B cells secreted only limited amounts of IgM antibodies and fail to produce IgG. In contrast the AID/YFP + IgM + B cells could give rise to new GL7 + B cells, suggesting that full reconstitution of the memory B cell pool may require transfer of the different antigen-experienced B cell subsets. Importantly, we found that the recall responses were more efficient if the transferred memory cells were given the required time to adapt to their new environment, suggesting that a period of accommodation is necessary before the transferred cells are fully capable to respond.", "Importantly, we found that the recall responses were more efficient if the transferred memory cells were given the required time to adapt to their new environment, suggesting that a period of accommodation is necessary before the transferred cells are fully capable to respond. Our findings also show that different processes can modify the survival conditions of memory B cells. Finally, we found that the generation of the memory B cell pool in response to an optimal dose of Ag did not rely on the number of the initially responding B cells, suggesting autonomous homeostatic controls for naïve and memory B cells a property that may allow reconstitution of the memory pool in immune-deficient hosts using a limited number of precursor naïve B cells.", "Finally, we found that the generation of the memory B cell pool in response to an optimal dose of Ag did not rely on the number of the initially responding B cells, suggesting autonomous homeostatic controls for naïve and memory B cells a property that may allow reconstitution of the memory pool in immune-deficient hosts using a limited number of precursor naïve B cells. An autonomous control of the memory B cell pool where each antigenic exposure adds new competing clones supports the notion of vaccination strategies using antigenic boosting to prevent memory B cell attrition. Overall the findings reported demonstrate that it is possible to reconstitute the memory B cell pool of an immune deficient host with an artificially induced population of monoclonal high affinity memory B cells." ]

[ "We investigated the ability of monoclonal B cells to restore primary and secondary T-cell dependent antibody responses in adoptive immune-deficient hosts. Priming induced B cell activation and expansion, AID expression, antibody production and the generation of IgM(+)IgG(-) and IgM(-)IgG(+) antigen-experienced B-cell subsets that persisted in the lymphopenic environment by cell division. Upon secondary transfer and recall the IgM(-)IgG(+) cells responded by the production of antigen-specific IgG while the IgM(+) memory cells secreted mainly IgM and little IgG, but generated new B cells expressing germinal center markers.", "Upon secondary transfer and recall the IgM(-)IgG(+) cells responded by the production of antigen-specific IgG while the IgM(+) memory cells secreted mainly IgM and little IgG, but generated new B cells expressing germinal center markers. The recall responses were more efficient if the antigenic boost was delayed suggesting that a period of adaptation is necessary before the transferred cells are able to respond. Overall these findings indicate that reconstitution of a functional and complete memory pool requires transfer of all different antigen-experienced B cell subsets.", "Overall these findings indicate that reconstitution of a functional and complete memory pool requires transfer of all different antigen-experienced B cell subsets. We also found that the size of the memory B cell pool did not rely on the number of the responding naïve B cells, suggesting autonomous homeostatic controls for naïve and memory B cells. By reconstituting a stable memory B cell pool in immune-deficient hosts using a monoclonal high-affinity B cell population we demonstrate the potential value of B cell adoptive immunotherapy.", "By reconstituting a stable memory B cell pool in immune-deficient hosts using a monoclonal high-affinity B cell population we demonstrate the potential value of B cell adoptive immunotherapy. Text: Immune responses to infectious agents have different out-comes that can either protect or fail to control disease. Protection from re-infection relies on the establishment of efficient secondary immune responses that require the generation of antigen-specific \"memory\" B and T lymphocytes.", "Protection from re-infection relies on the establishment of efficient secondary immune responses that require the generation of antigen-specific \"memory\" B and T lymphocytes. The generation and selection of T-cell dependent \"memory\" B cells involves distinct molecular mechanisms: immunoglobulin isotype recombination and somatic hyper mutation, both dependent on the expression of AID [1] . Therefore, a long-standing paradigm defined memory B cells as IgM -IgG + isotype switched cells [2] .", "Therefore, a long-standing paradigm defined memory B cells as IgM -IgG + isotype switched cells [2] . Different lines of evidence indicate that this is not always the case. In humans, it has been shown that some IgM + B cells bear the phenotype of other memory cells, being CD27 + , and carry frequent point mutations in the V region of the Ig genes, suggesting that they must represent highly selected B cell populations [3] .", "In humans, it has been shown that some IgM + B cells bear the phenotype of other memory cells, being CD27 + , and carry frequent point mutations in the V region of the Ig genes, suggesting that they must represent highly selected B cell populations [3] . In mice, populations of CD19 + IgM + able to mount secondary responses have been identified [4] [5] [6] [7] . Overall these findings suggest that the T-cell dependent memory B cell pool comprises distinct subsets of memory B cells with different properties and effector functions [4] [5] [6] .", "Overall these findings suggest that the T-cell dependent memory B cell pool comprises distinct subsets of memory B cells with different properties and effector functions [4] [5] [6] . The biological properties that ensure the long-term persistence of memory and efficient secondary antibody responses have not been yet completely established. While initial studies proposed that after transfer memory B cells faded rapidly [8, 9] suggesting that long-lasting memory required the continuous recruitment of new cells [8] and/or antigen persistence [9, 10] , others suggested that memory B cells were able of extended survival without cell division [11] in the absence of antigen [2] .", "While initial studies proposed that after transfer memory B cells faded rapidly [8, 9] suggesting that long-lasting memory required the continuous recruitment of new cells [8] and/or antigen persistence [9, 10] , others suggested that memory B cells were able of extended survival without cell division [11] in the absence of antigen [2] . Long-term persistence of antibody responses has also been attributed to populations of long-lived plasma cells mainly resident in the bone marrow following immunization [12, 13] . The demonstration of the compartmentalization of \"antibody memory\" into different cellular layers suggested that the separate subsets of memory B cells behave differently.", "The demonstration of the compartmentalization of \"antibody memory\" into different cellular layers suggested that the separate subsets of memory B cells behave differently. Accordingly, it has been reported that IgG + cells that could rapidly respond upon challenge did not persist long, while IgM + cells could generate a second wave of germinal center responses allowing persistence of memory [4] [5] [6] 14] . Currently, immunotherapy approaches using passive antibody transfer [15, 16] ) is limited by the short half-life of immunoglobulin.", "Currently, immunotherapy approaches using passive antibody transfer [15, 16] ) is limited by the short half-life of immunoglobulin. Therefore new therapy strategies may require the adoptive transfer of high-affinity memory B cells, ready to respond and able to persist. The development of these new strategies requires a profound understanding of the mechanisms that regulate memory B cell numbers and ensure long persistence upon adoptive transfer.", "The development of these new strategies requires a profound understanding of the mechanisms that regulate memory B cell numbers and ensure long persistence upon adoptive transfer. Moreover, knowledge of the mechanisms that determine the size of the memory B cell pool may be also critical to device new reconstitution strategies. So far, studies comparing populations of naïve and memory B cells have been hindered both by the vast clonal heterogeneity of the cells involved and by our inability to generate significant numbers of antigen specific memory B cells.", "So far, studies comparing populations of naïve and memory B cells have been hindered both by the vast clonal heterogeneity of the cells involved and by our inability to generate significant numbers of antigen specific memory B cells. Indeed in a normal laboratory mouse the population of B cells bearing a \"memory IgG + phenotype' represent a small fraction of the total B cell pool (<0.5%) and upon immunization the number of the clonal diverse antigen-specific memory B cells generated is generally very limited (<10 3 ) [1, 6] . To circumvent these limits, we decided to compare the properties of homogeneous populations of naïve and memory B cells of known antigen specificity, belonging to the same clone.", "To circumvent these limits, we decided to compare the properties of homogeneous populations of naïve and memory B cells of known antigen specificity, belonging to the same clone. We used SW HEL transgenic mice where B cells bear a high-affinity BCR specific for HEL and are capable of class switch recombination and somatic hypermutation (SHM) [17, 18] . To identify \"memory B cells\" the SW HEL mice were crossed with mice where AID transcription provokes the permanent expression of an YFP reporter in post-germinal center lymphocytes [19] .", "To identify \"memory B cells\" the SW HEL mice were crossed with mice where AID transcription provokes the permanent expression of an YFP reporter in post-germinal center lymphocytes [19] . These mice were in a Rag2-deficient background and therefore contain a pure population of monoclonal HEL-specific B cells. To generate memory cells, purified naïve B cells from the SW HEL .AID/YFP.Rag2 -/mice were transferred into adoptive hosts together with monoclonal OVA-specific CD4 + T cells from OTII.Rag2 -/-TCR transgenic mice.", "To generate memory cells, purified naïve B cells from the SW HEL .AID/YFP.Rag2 -/mice were transferred into adoptive hosts together with monoclonal OVA-specific CD4 + T cells from OTII.Rag2 -/-TCR transgenic mice. Upon immunization with OVA-HEL complexes, we obtained a significant number of persisting HEL-specific IgM + Ig-G -YFP + and IgM -IgG + YFP + memory B cells, number that did not correlate to the number of precursor naïve cells initially injected suggesting that the memory B cell pool is regulated independently. We characterized the functional capacity of these two memory cell types in immune deficient hosts.", "We characterized the functional capacity of these two memory cell types in immune deficient hosts. Mice B6 and B6.Rag2-/- [20] mice were kept at the Centre Des Techniques Avancées (CDTA), Centre National de la Recherche Scientifique (CNRS), Orleans, France; SWHEL.AID/YFP.Rag-/-mice, obtained by crossing SWHEL (18)(a gift of Dr. Robert Brink) and AID/YFP [19] (a gift of Dr. Rafael Casellas) with B6.Rag2-/-mice. OTII.Rag-/-mice were kept in our animal facilities at the Pasteur Institute.", "OTII.Rag-/-mice were kept in our animal facilities at the Pasteur Institute. Experiments were preformed according to Pasteur Institute Safety Committee in accordance with French and European guidelines and the ethics Committee of Paris 1 (permits 2010-0002, -0003 and -0004). Euthanasia of the mice was performed by cervical dislocation.", "Euthanasia of the mice was performed by cervical dislocation. This specific study was approved by the European Research Council (ERC) committee related to the grant AdG09 249740-QSIS. The general status of the mice was controlled daily by monitoring the appearence of obvious pain, distress or suffering (prostration, respiratory issues, loss of weight).", "The general status of the mice was controlled daily by monitoring the appearence of obvious pain, distress or suffering (prostration, respiratory issues, loss of weight). The end-point of the experiment was determined by a loss of more than 20% of the weight or as soon as the distress signs appeared. In this case, experiment was stopped and the animals were euthanized.", "In this case, experiment was stopped and the animals were euthanized. Single-cell suspensions of B cells from spleens and lymph nodes of SW HEL .AID/YFP.Rag -/mice together with CD4 + T cells from spleens and lymph nodes of OTII.Rag -/mice were transferred intravenously into the retro-orbital sinus of B6.Ly5 a IgH a or B6.Rag2 -/recipient mice. Mice received 10 6 HEL + B cells and 10 6 CD4 + T cells unless stated otherwise.", "Mice received 10 6 HEL + B cells and 10 6 CD4 + T cells unless stated otherwise. Mice were immunized 24H later with 1 mg of Ovalbumin coupled to Hen Egg Lysozyme (OVA-HEL) in 50μg of Alu-S-Gel (Serva) we determined as the optimal dose of Ag (data not shown). Naive cells from SW HEL .AID/YFP.Rag -/mice and memory B cells subsets from immunized B6.Rag -/hosts mice were purified from spleens and lymph nodes by flow cytometry sorting.", "Naive cells from SW HEL .AID/YFP.Rag -/mice and memory B cells subsets from immunized B6.Rag -/hosts mice were purified from spleens and lymph nodes by flow cytometry sorting. Single-cell suspensions containing 5×10 4 B cells and 10 6 T cells were transferred intravenously into B6.Rag2 -/recipient hosts. The purity of sorted cells was above 98%.", "The purity of sorted cells was above 98%. 24 h after transfer, mice were immunized with 1 mg of OVA-HEL. Spleen, bone marrow, inguinal and mesenteric lymph nodes single-cell suspensions were stained for cell surface or intracellular proteins with appropriate combinations of the following monoclonal antibodies conjugated to pacific blue, Qdot-655, Brillant Violet 605, allophycocyanin, peridinin chlorophyll protein-cyanine 5.5, phycoerythrin, phycoerythrin-cyanine7: anti-CD19 (6D5), anti-IgM (R6-60.2), anti-IgG1 (X56), anti-CD138 (281-2), anti-Gl7 (Gl7), anti-CD95 (Jo2), anti-CD62L (MEL-14), anti-CD69 (H1-2F3), anti-BAFFR (7H22-E16), anti-CXCR5 (L138D7), anti-IA b (AF6-120.1), anti-CD80 (16-10A1), anti-CD73 (TY-11-8) and anti-PDL2 (TY25) and anti-Ki-67 (mm1) purchased from Becton Dickinson Pharmingen, Biolegend, Invitrogen and eBioscience.", "Spleen, bone marrow, inguinal and mesenteric lymph nodes single-cell suspensions were stained for cell surface or intracellular proteins with appropriate combinations of the following monoclonal antibodies conjugated to pacific blue, Qdot-655, Brillant Violet 605, allophycocyanin, peridinin chlorophyll protein-cyanine 5.5, phycoerythrin, phycoerythrin-cyanine7: anti-CD19 (6D5), anti-IgM (R6-60.2), anti-IgG1 (X56), anti-CD138 (281-2), anti-Gl7 (Gl7), anti-CD95 (Jo2), anti-CD62L (MEL-14), anti-CD69 (H1-2F3), anti-BAFFR (7H22-E16), anti-CXCR5 (L138D7), anti-IA b (AF6-120.1), anti-CD80 (16-10A1), anti-CD73 (TY-11-8) and anti-PDL2 (TY25) and anti-Ki-67 (mm1) purchased from Becton Dickinson Pharmingen, Biolegend, Invitrogen and eBioscience. Cells were also stained with HEL (Sigma) coupled with AF594 using Alexa Fluor1 594 Protein Labeling Kit from Life technologies. Before staining, cells were treated with Fc-Block (CD16/CD32, Becton Dickinson Pharmingen).", "Before staining, cells were treated with Fc-Block (CD16/CD32, Becton Dickinson Pharmingen). Dead cells were excluded during analysis according to their light-scattering characteristics. For intracellular stainings, cells were first stained with antibodies specific for cell surface antigens. Then, cells were fixed and permeabilized according the manufacturer's recommendations (BD Bisciences).", "Then, cells were fixed and permeabilized according the manufacturer's recommendations (BD Bisciences). For proliferation assay, mice were injected i.p. with 50 mg/kg of BrdU (Sigma-Aldrich) and were killed 24 or 72 hours later. Incorporated BrdU was detected intracellularly using anti-BrdU APC-conjugated antibodies according to the manufacturer's recommendations (BD Biosciences).", "Incorporated BrdU was detected intracellularly using anti-BrdU APC-conjugated antibodies according to the manufacturer's recommendations (BD Biosciences). All data acquisitions and analyses were performed with LSRFortessa (Becton Dickinson) interfaced with BD FACSDiva (Becton Dickinson) and FlowJo (Tree Star) software. Subsets of memory B cells were sorted as CD19 + HEL + YFP + IgM + or IgG + and naive cells as CD19 + HEL + YFP -IgM + using a FACSAriaIII flow cytometer.", "Subsets of memory B cells were sorted as CD19 + HEL + YFP + IgM + or IgG + and naive cells as CD19 + HEL + YFP -IgM + using a FACSAriaIII flow cytometer. The purity of the sorted populations varied from 90-95%. Sera HEL-specific Ig concentrations were quantified by ELISA.", "Sera HEL-specific Ig concentrations were quantified by ELISA. Plates were coated with HEL and saturated with PBS-5% Milk. Dilutions of sera were added. After incubation (2 hours, 37˚C) and washing, HRP-labeled anti-mouse IgM or IgG antibodies were added. After incubation and washing, bound antibodies were revealed with the substrate O-phenylenediamine and H2O2.", "After incubation and washing, bound antibodies were revealed with the substrate O-phenylenediamine and H2O2. The reaction was stopped after 10 min. by addition of 10% SDS and the absorbance read at 492nm in a multiscan spectrometer.", "by addition of 10% SDS and the absorbance read at 492nm in a multiscan spectrometer. Ig concentrations were determined by comparing the displacement of the dilution curves in the linear interval between standards at a concentration of 1 mg/ml and the serum samples. The quantification of IgG or IgM secreting cells was assayed by ELISpot technique.", "The quantification of IgG or IgM secreting cells was assayed by ELISpot technique. Briefly, plates were coated with HEL. After saturating, the cells were distributed into the micro wells in RPMI1640-2%FCS. The plates were incubated for 12 h at 37˚C, 5% CO2 atmosphere.", "The plates were incubated for 12 h at 37˚C, 5% CO2 atmosphere. After extensive wash, plates were incubated with goat anti-mouse IgM or anti-IgG labeled with alkaline phosphatase. After washing, the revealing substrate was added (2,3 mM 5-bromo-4-chloro-3-indolyl phosphate diluted in 2-amino-2-methyl-1-proprenolol buffer).", "After washing, the revealing substrate was added (2,3 mM 5-bromo-4-chloro-3-indolyl phosphate diluted in 2-amino-2-methyl-1-proprenolol buffer). Spleens from 14 day-immunized mice were initially fixed with paraformaldehyde and embedded in 4% low-gelling-temperature agarose (type VII-A; Sigma-Aldrich) prepared in PBS. 150μm slices were cut with a vibratome (VT 1000S; Leica) in a bath of ice-cold PBS.", "150μm slices were cut with a vibratome (VT 1000S; Leica) in a bath of ice-cold PBS. For immunolabeling, samples were saturated with PBS supplemented with 10% of fetal calf serum, then were labeled with primary antibodies anti-B220-APC (clone RA3-6B2) and anti-IgD-PE (clone 11-26c.2a) and analyzed with a spinning disk confocal microscope equipped with a CoolSnap HQ2 camera (Photometrics) and a 20x objective. Images were acquired and analyzed with MetaMorph 7 imaging software Molecular Devices).", "Images were acquired and analyzed with MetaMorph 7 imaging software Molecular Devices). Sample means were compared using the Student's t test. Sample means were considered significantly different at p < 0.05.", "Sample means were considered significantly different at p < 0.05. During an immune response the complexity of determinants expressed by immunizing antigen and the degeneracy of antigen-specific recognition results in a vast heterogeneity of responding cells rendering impossible the direct comparison of the properties of naïve and memory B cells belonging to the same clone. We have devised an experimental system that permits the comparison between naïve and memory B cells expressing the same antigen receptor and allows marking permanently memory B cells.", "We have devised an experimental system that permits the comparison between naïve and memory B cells expressing the same antigen receptor and allows marking permanently memory B cells. For that purpose we used SW HEL transgenic mice in a Rag2-deficient background holding a single population of monoclonal B cells, all bearing a high-affinity BCR specific for HEL and capable of class switch recombination and somatic hypermutation (SHM) [17, 18] . To identify antigen-experienced B cells the SW HEL .", "To identify antigen-experienced B cells the SW HEL . Rag2 -/mice were crossed with mice where AID transcription induces the permanent expression of an YFP reporter in post-germinal center lymphocytes [19] . Since in intact Tg mice immune responses were not traceable, probably because of the presence of low level pre-existing anti-HEL antibodies that neutralize the immunizing protein, we used an adoptive cell transfer strategy to study the ability of the high affinity monoclonal B cell to reconstitute response in immune-deficient hosts and generate antibody memory.", "Since in intact Tg mice immune responses were not traceable, probably because of the presence of low level pre-existing anti-HEL antibodies that neutralize the immunizing protein, we used an adoptive cell transfer strategy to study the ability of the high affinity monoclonal B cell to reconstitute response in immune-deficient hosts and generate antibody memory. Purified naïve B cells from the SW HEL .AID/YFP.Rag2 -/mice were transferred into Rag2-deficient mice together with monoclonal OVA-specific CD4 + T helper cells from OTII.Rag2 -/-TCR transgenic mice. The day after, host mice were immunized with OVA-HEL complexes (Fig 1A) .", "The day after, host mice were immunized with OVA-HEL complexes (Fig 1A) . In these conditions, antigenic challenge resulted in B cell activation and the development of significant numbers of CD19 + HEL + AID/YFP + B cells, which were not detected in non-immunized mice or in mice immunized in absence of helper T cells (Fig 1B) . We followed the early kinetics of this response.", "We followed the early kinetics of this response. The number of HEL-specific B cells increased from the initial 2x10 6 transferred to about 15x10 6 at day 14 (Fig 1C left) the B cells expressing AID/YFP being the dominant population (Fig 1C right) . A fraction of the HEL-specific B cells underwent class switch recombination and at day 14 we recovered both IgM + IgG -AID/YFP + and IgM -IgG + AID/YFP + cell populations (Fig 1B) .", "A fraction of the HEL-specific B cells underwent class switch recombination and at day 14 we recovered both IgM + IgG -AID/YFP + and IgM -IgG + AID/YFP + cell populations (Fig 1B) . B cell expansion and phenotypic changes were accompanied by the production of IgM and IgG HEL-specific antibodies ( Fig 1D) . Two weeks after antigenic challenge we observed the formation of germinal centers in the spleen of the host mice ( Fig 1E) .", "Two weeks after antigenic challenge we observed the formation of germinal centers in the spleen of the host mice ( Fig 1E) . Coherently we found that while upon adoptive transfer all B cells expressed CD95, only after antigenic challenge most YFP + B cells expressed the germinal center specific marker GL7 (Fig 1F) . In conclusion, the adoptive cell transfer strategy allowed the development of a primary immune response with B cell activation and expansion, induction of AID expression, class switch recombination, antigen-specific IgM and IgG antibody production and germinal center formation.", "In conclusion, the adoptive cell transfer strategy allowed the development of a primary immune response with B cell activation and expansion, induction of AID expression, class switch recombination, antigen-specific IgM and IgG antibody production and germinal center formation. We studied the evolution of the B cell response. From two weeks onwards the total number of B cells contracted and at four weeks we recovered about 2-4x10 6 cells, number that remained stable up to week 20 (Fig 2A) .", "From two weeks onwards the total number of B cells contracted and at four weeks we recovered about 2-4x10 6 cells, number that remained stable up to week 20 (Fig 2A) . High titers of HEL-specific IgG were kept from week 3 to 8, declined thereafter, but were still significantly elevated 20 weeks later (Fig 2B) . A population of cells secreting HEL-specific Igs was present in the spleen (Fig 2C) , but not in the BM (not shown) even at the late time points.", "A population of cells secreting HEL-specific Igs was present in the spleen (Fig 2C) , but not in the BM (not shown) even at the late time points. About 60% of the recovered cells exhibited the phenotype of antigen-experienced (\"memory\") CD19 + HEL + AID/YFP + expressing either IgM or IgG ( Fig 2D and 2E) . We compared the phenotype of the two AID/YFP + IgM + and AID/YFP + IgM -IgG + memory cell populations recovered with that of the naïve B cells (Fig 2F) .", "We compared the phenotype of the two AID/YFP + IgM + and AID/YFP + IgM -IgG + memory cell populations recovered with that of the naïve B cells (Fig 2F) . We found that antigen-experience and naïve B cells expressed similar levels of CD62L, CD69 and BAFFR (not shown). Antigen-experienced cells presented sustained expression of CD95 and increased levels of PNA, but the vast majority lost expression of the germinal center marker GL7 present at earlier times post-immunization ( Fig 2F compare to Fig 1F) .", "Antigen-experienced cells presented sustained expression of CD95 and increased levels of PNA, but the vast majority lost expression of the germinal center marker GL7 present at earlier times post-immunization ( Fig 2F compare to Fig 1F) . Compared to naïve B cells, AID/ YFP + cells expressed higher levels of CD80 and MHC class II and down-regulated expression of CXCR5 (Fig 2F) . These findings indicate that the post-germinal center AID/YFP + B cells express an activated phenotype [5, 21] , have increased antigen-presenting capacity [22] , but may loose the ability to re-enter primary follicles being CXCR5 low [23] .", "These findings indicate that the post-germinal center AID/YFP + B cells express an activated phenotype [5, 21] , have increased antigen-presenting capacity [22] , but may loose the ability to re-enter primary follicles being CXCR5 low [23] . We have also compared the patterns of gene expression (RNAseq) by naïve, activated (YFPcells of immunized mice) and both populations of YFP + memory cells. The data shows a clear discrimination of naïve and activated/memory cells while indicating only minor differences between both subsets of YFP + memory cells (Fig 3) .", "The data shows a clear discrimination of naïve and activated/memory cells while indicating only minor differences between both subsets of YFP + memory cells (Fig 3) . mRNA was isolated from sort-purified Naïve (CD19 + HEL + YFP -IgM + ) IgM + IgGor IgM -IgG + HEL + CD19 + YFP + memory B cells from spleen of different recipient mice. Total recommended by the manufacturer.", "Total recommended by the manufacturer. The validated libraries were then subjected to DNA sequencing. The analysis is performed using the R software, Bioconductor packages including DESeq2 and the PF2tools package (version 1.2.9) developed at PF2 (Institut Pasteur).", "The analysis is performed using the R software, Bioconductor packages including DESeq2 and the PF2tools package (version 1.2.9) developed at PF2 (Institut Pasteur). Normalization and differential analysis are carried out according to the DESeq2 model and package (version 1.8.1). Fig 3A shows a representative heat map of the different cells populations.", "Fig 3A shows a representative heat map of the different cells populations. Fig 3B shows \n\nLate in the immune response persistent B cell numbers were kept by active cell division as a significant fraction of the cells were Ki67 + (Fig 2G left) and incorporated BrdU (Fig 2G middle) . The frequency of BrdU + cells was higher among the AID/YFPcells (15%) than in the major AID/YFP + memory population (3%) and similar between the IgM + and IgM -AID/ YFP + populations ( Fig 2G middle and not shown) .", "The frequency of BrdU + cells was higher among the AID/YFPcells (15%) than in the major AID/YFP + memory population (3%) and similar between the IgM + and IgM -AID/ YFP + populations ( Fig 2G middle and not shown) . Three days after BrdU pulse populations were clear of BrdU + cells (Fig 2G right) attesting their high division rate. In spite of their increased proliferation rate, memory cells numbers were stable indicating that proliferation may be compensated by cell death as suggested by the frequency of caspase3 + cells (Fig 2H) .", "In spite of their increased proliferation rate, memory cells numbers were stable indicating that proliferation may be compensated by cell death as suggested by the frequency of caspase3 + cells (Fig 2H) . The frequency of Caspase3 + cells was higher among the AID/YFP + cells suggesting that a fraction of these cells may represent cells undergoing terminal differentiation. Importantly, these findings demonstrate that the transfer strategy allowed the generation of significant numbers of persisting antigen-experienced YFP + cells.", "Importantly, these findings demonstrate that the transfer strategy allowed the generation of significant numbers of persisting antigen-experienced YFP + cells. It is not yet known whether the number of antigen-experienced memory B cells correlated to the number of naïve B cells or if it is controlled independently of the initial number of antigenspecific B cells present. To approach this question we transferred different numbers of mature naïve B cells from SW HEL .AID/YFP.Rag2 -/donors (ranging from 10 5 to 5.10 6 ) into Rag2-deficient mice together with an excess of CD4 + T helper cells (10 6 ) and immunize the hosts the day after cell transfer with OVA-HEL in optimal non-limiting quantities.", "To approach this question we transferred different numbers of mature naïve B cells from SW HEL .AID/YFP.Rag2 -/donors (ranging from 10 5 to 5.10 6 ) into Rag2-deficient mice together with an excess of CD4 + T helper cells (10 6 ) and immunize the hosts the day after cell transfer with OVA-HEL in optimal non-limiting quantities. To directly compare the results obtained after the transfer of different all numbers we allowed the responses to reach steady-state eight weeks after antigenic challenge. We studied the amplitude of the immune response by measuring the serum titers of HEL-specific IgG antibodies and enumerating the number of HEL-specific B cells recovered.", "We studied the amplitude of the immune response by measuring the serum titers of HEL-specific IgG antibodies and enumerating the number of HEL-specific B cells recovered. We found that in the presence of excess T cell help, the levels of the HEL-specific IgGs (Fig 4C) , and both the total number of HEL-specific ( Fig 4A) and of memory YFP + B cells recovered (Fig 4B) , did not correlate to the number of antigen specific naïve B cells initially transferred. Memory B cells are defined functionally by their ability to induce secondary IgG antibody responses upon secondary antigenic challenge.", "Memory B cells are defined functionally by their ability to induce secondary IgG antibody responses upon secondary antigenic challenge. We investigated whether the subsets of AID/ YFP + IgM + and AID/YFP + IgM -IgG + antigen-experienced (memory) B cells persisting at late time points could mount secondary IgG responses and persist after secondary transfer. For this purpose we followed two different experimental strategies.", "For this purpose we followed two different experimental strategies. In the first, 5x10 4 cells of either IgM + or IgM -IgG + memory B cells, were transferred with an excess helper OTII CD4 + T cells into secondary Rag-deficient hosts that were boosted with OVA-HEL the day after cell transfer. In the absence of immunization antibody levels were undetectable (not shown) and three weeks after transfer recovery of both memory B cell subsets was about 10-20% of the initial cell input, exceeding naïve B cell recovery (Fig 5A) , supporting the notion that memory B cells may not require specific ligand recognition to survive (2).", "In the absence of immunization antibody levels were undetectable (not shown) and three weeks after transfer recovery of both memory B cell subsets was about 10-20% of the initial cell input, exceeding naïve B cell recovery (Fig 5A) , supporting the notion that memory B cells may not require specific ligand recognition to survive (2). One cannot exclude, however, that cross-reactivity of the BCR transgene with environmental antigens may allow signaling sufficient to maintain naïve and memory cell survival in the absence of HEL [24] . Following immunization, the secondarily transferred AID/YFP + IgM -IgG + cells responded promptly with the exclusive production of significant levels HEL-specific IgG thus confirming their memory statute (11) .", "Following immunization, the secondarily transferred AID/YFP + IgM -IgG + cells responded promptly with the exclusive production of significant levels HEL-specific IgG thus confirming their memory statute (11) . The AID/YFP + IgM + B cells in response to antigenic boost produced only limited amounts of IgM antibodies (Fig 4B) , little IgG antibodies, but did generate GL7 + B cells more efficiently than the IgG + memory B cell population (Fig 5D) . Thus the IgM + subset may contain precursors able to generate a secondary germinal center reaction and a new progeny of IgG + effectors (4).", "Thus the IgM + subset may contain precursors able to generate a secondary germinal center reaction and a new progeny of IgG + effectors (4). With time antibody levels decayed rapidly suggesting that the number of transferred memory B cells declined in the secondary hosts after antigenic boost. Indeed, IgM + and IgG + memory B cells failed to expand and 3 weeks after immunization cell recovery was similar to the retrieval observed in the non-immunized hosts (compare Fig 5E and 5A) .", "Indeed, IgM + and IgG + memory B cells failed to expand and 3 weeks after immunization cell recovery was similar to the retrieval observed in the non-immunized hosts (compare Fig 5E and 5A) . In similar experimental conditions, naïve B cells following immunization expanded, acquired AID/ YFP expression and their numbers more than doubled the number initially injected (Figs 5F and 2A). These data suggest that a significant fraction of the memory B cells generated have a reduced expansion capacity being programmed for rapid differentiation for effector functions.", "These data suggest that a significant fraction of the memory B cells generated have a reduced expansion capacity being programmed for rapid differentiation for effector functions. Besides long-term survival memory B cells must maintain functional activity in the absence of nominal antigen to be fully effective. To test this we used an alternative approach where memory cells were parked in secondary Rag-deficient hosts for 30 days before re-immunization.", "To test this we used an alternative approach where memory cells were parked in secondary Rag-deficient hosts for 30 days before re-immunization. We found that under these conditions antigenic challenge resulted in the production of HEL-specific IgG antibodies and in a 100 fold increase in the number of cells recovered, expansion that largely exceed that observed after immediate challenge (Fig 5G) . The aim of this study was to characterize the fate of activated B cells and the generation of memory B cells.", "The aim of this study was to characterize the fate of activated B cells and the generation of memory B cells. To do this, we adoptively transferred monoclonal B cells into immune deficient hosts followed by immunization in presence of T cell help. This strategy resulted in the development of different B cell memory subsets, namely IgM + and IgG + , as described for in situ generated memory cells [4, 6, 14] .", "This strategy resulted in the development of different B cell memory subsets, namely IgM + and IgG + , as described for in situ generated memory cells [4, 6, 14] . These findings indicate that distinct memory B cell subsets are not the result of the heterogeneity of initially responding naive cells, but originate from the differentiation of a single B cell clone. While studying the respective rate of proliferation of both types of memory B cells, we found the same high rate of proliferation for IgM + and IgG + memory B cells.", "While studying the respective rate of proliferation of both types of memory B cells, we found the same high rate of proliferation for IgM + and IgG + memory B cells. These results contrast with previous published data. First it was been reported that \"in situ\" memory B cells persist as resting non-dividing cells [11, 25] .", "First it was been reported that \"in situ\" memory B cells persist as resting non-dividing cells [11, 25] . However, we have shown that upon adoptive transfer and in absence of competing cells, B cells increase their division rate to occupy the available empty niche [26] , which may explain the higher division rate observed here using this adoptive cell transfer strategy. Secondly, comparing life spans among heterogeneous memory B cell populations it was previously reported a lower division rate among the IgM + subset compared to the IgG + polyclonal subset [6] .", "Secondly, comparing life spans among heterogeneous memory B cell populations it was previously reported a lower division rate among the IgM + subset compared to the IgG + polyclonal subset [6] . Differences in BCR affinity between IgM + and IgG + memory clones may explain the higher division rate previously observed among the IgG + cells [6] . In contrast we compared memory B cell subsets belonging to the same clone bearing the same high affinity BCR.", "In contrast we compared memory B cell subsets belonging to the same clone bearing the same high affinity BCR. Overall these observations support the notion that lymphocyte division rates and life spans are not an intrinsic cell property, but rather determined by the environment and the presence of competing populations [27] . They demonstrate that upon the correct conditions memory B cells can persist by cell division.", "They demonstrate that upon the correct conditions memory B cells can persist by cell division. An important question was whether the number of memory B cells depends on the number of initial naïve B cells. We found that, in the presence of an excess of T cell help, that was not the case.", "We found that, in the presence of an excess of T cell help, that was not the case. However, it was previously reported during polyclonal responses that serum titers of anti-HSA was proportional to the number of cells transferred into irradiated mice [28] . It is possible that limited antigen-specific T-B cell encounters may constraint the number of responding B cells and thus determine linear precursor-progeny between naïve and memory B cells.", "It is possible that limited antigen-specific T-B cell encounters may constraint the number of responding B cells and thus determine linear precursor-progeny between naïve and memory B cells. Our findings indicate that within a single clone the number of precursor naive B cells present in the peripheral B cell pool does determine neither the intensity nor the final number of memory B cells in response to an optimal dose of antigen. They suggest that the size of memory B cell pool may be controlled independently of the number of naïve B cell precursors and that in the absence of clonal competition the memory niche can be filled with a single monoclonal population.", "They suggest that the size of memory B cell pool may be controlled independently of the number of naïve B cell precursors and that in the absence of clonal competition the memory niche can be filled with a single monoclonal population. Considering diverse polyclonal populations, the limited niche for memory cells will imply strong competition among clones resulting in the selection of best fit (high affinity) cells: rare mutated clones being able to out compete more frequent but less avid clones. In our settings, the transgenic memory B cells are likely to counter select any new mutant clones since they express a very high affinity BCR selected in the course of a secondary immune response [29] .", "In our settings, the transgenic memory B cells are likely to counter select any new mutant clones since they express a very high affinity BCR selected in the course of a secondary immune response [29] . Thus, notwithstanding the expression of AID and proliferation we did not detect any BCR VH and VL Ig-chain nucleotide mutations among the recovered memory B cells (not shown). These findings may have implication for vaccination protocols as they indicate that each new antigenic exposure or unrelated immunization would add extra competing clones supporting the need for repeated antigenic boosts to prevent memory B cell attrition.", "These findings may have implication for vaccination protocols as they indicate that each new antigenic exposure or unrelated immunization would add extra competing clones supporting the need for repeated antigenic boosts to prevent memory B cell attrition. They also demonstrate that the memory B cell pool can be reconstituted from a relatively small number of antigen-specific cells. It is likely that the relatively poor memory B cell expansion observed after immediate boost after adoptive transfer could be due to the lack in Rag-deficient hosts of the appropriate environment required for memory B cell survival and function.", "It is likely that the relatively poor memory B cell expansion observed after immediate boost after adoptive transfer could be due to the lack in Rag-deficient hosts of the appropriate environment required for memory B cell survival and function. It should be pointed out that B cell transfer into transgenic ML5 Rag-deficient hosts expressing low levels of HEL [29] resulted in rapid cell loss and recovery suggesting that in these hosts, B cells are trapped by antigen in locations were they are unable to survive (not shown). Nevertheless, it has been shown that B cells can drive the maturation of follicular dendritic cells and the organization of lymphoid follicles [30] .", "Nevertheless, it has been shown that B cells can drive the maturation of follicular dendritic cells and the organization of lymphoid follicles [30] . Similarly, transferred helper cells may also modify their immediate environment. Thus, by allowing lymphocytes to adapt and modify their immediate environment we improved their response and more important, we recovered the memory B cell pool size present in the original donor mice.", "Thus, by allowing lymphocytes to adapt and modify their immediate environment we improved their response and more important, we recovered the memory B cell pool size present in the original donor mice. In this study we show that it is possible to fully reconstitute a primary response and the establishment of antibody memory in immune deficient mice after adoptive transfer of antigen-specific monoclonal B cells together with a population of monoclonal helper T cells. Indeed, it is generally believed that in immune deficiencies, B cell therapy has restricted application due to intrinsic defects of host's lymphoid organs structure that may prevent development of immune responses, germinal center formation, establishment of antibody memory and limit cell survival.", "Indeed, it is generally believed that in immune deficiencies, B cell therapy has restricted application due to intrinsic defects of host's lymphoid organs structure that may prevent development of immune responses, germinal center formation, establishment of antibody memory and limit cell survival. In contrast we showed that after adoptive transfer in immune deficient hosts antigen immunization induced B cell activation and expansion, induction of AID expression, class switch recombination, antigen-specific IgM and IgG antibody production, germinal center formation and the generation of two subsets of AID/YFP + IgM + IgGand AID/YFP + Ig-M -IgG + antigen-experienced B cell subsets able to persist in a lymphopenic environment by cell division mimicking responses obtained in intact non-Tg mice [4] . Upon challenge the AID/YFP + IgM -IgG + cells responded promptly with the production of HEL-specific IgG while the AID/YFP + IgM + B cells secreted only limited amounts of IgM antibodies and fail to produce IgG.", "Upon challenge the AID/YFP + IgM -IgG + cells responded promptly with the production of HEL-specific IgG while the AID/YFP + IgM + B cells secreted only limited amounts of IgM antibodies and fail to produce IgG. In contrast the AID/YFP + IgM + B cells could give rise to new GL7 + B cells, suggesting that full reconstitution of the memory B cell pool may require transfer of the different antigen-experienced B cell subsets. Importantly, we found that the recall responses were more efficient if the transferred memory cells were given the required time to adapt to their new environment, suggesting that a period of accommodation is necessary before the transferred cells are fully capable to respond.", "Importantly, we found that the recall responses were more efficient if the transferred memory cells were given the required time to adapt to their new environment, suggesting that a period of accommodation is necessary before the transferred cells are fully capable to respond. Our findings also show that different processes can modify the survival conditions of memory B cells. Finally, we found that the generation of the memory B cell pool in response to an optimal dose of Ag did not rely on the number of the initially responding B cells, suggesting autonomous homeostatic controls for naïve and memory B cells a property that may allow reconstitution of the memory pool in immune-deficient hosts using a limited number of precursor naïve B cells.", "Finally, we found that the generation of the memory B cell pool in response to an optimal dose of Ag did not rely on the number of the initially responding B cells, suggesting autonomous homeostatic controls for naïve and memory B cells a property that may allow reconstitution of the memory pool in immune-deficient hosts using a limited number of precursor naïve B cells. An autonomous control of the memory B cell pool where each antigenic exposure adds new competing clones supports the notion of vaccination strategies using antigenic boosting to prevent memory B cell attrition. Overall the findings reported demonstrate that it is possible to reconstitute the memory B cell pool of an immune deficient host with an artificially induced population of monoclonal high affinity memory B cells." ]

[ "We investigated the ability of monoclonal B cells to restore primary and secondary T-cell dependent antibody responses in adoptive immune-deficient hosts. Priming induced B cell activation and expansion, AID expression, antibody production and the generation of IgM(+)IgG(-) and IgM(-)IgG(+) antigen-experienced B-cell subsets that persisted in the lymphopenic environment by cell division. Upon secondary transfer and recall the IgM(-)IgG(+) cells responded by the production of antigen-specific IgG while the IgM(+) memory cells secreted mainly IgM and little IgG, but generated new B cells expressing germinal center markers.", "Upon secondary transfer and recall the IgM(-)IgG(+) cells responded by the production of antigen-specific IgG while the IgM(+) memory cells secreted mainly IgM and little IgG, but generated new B cells expressing germinal center markers. The recall responses were more efficient if the antigenic boost was delayed suggesting that a period of adaptation is necessary before the transferred cells are able to respond. Overall these findings indicate that reconstitution of a functional and complete memory pool requires transfer of all different antigen-experienced B cell subsets.", "Overall these findings indicate that reconstitution of a functional and complete memory pool requires transfer of all different antigen-experienced B cell subsets. We also found that the size of the memory B cell pool did not rely on the number of the responding naïve B cells, suggesting autonomous homeostatic controls for naïve and memory B cells. By reconstituting a stable memory B cell pool in immune-deficient hosts using a monoclonal high-affinity B cell population we demonstrate the potential value of B cell adoptive immunotherapy.", "By reconstituting a stable memory B cell pool in immune-deficient hosts using a monoclonal high-affinity B cell population we demonstrate the potential value of B cell adoptive immunotherapy. Text: Immune responses to infectious agents have different out-comes that can either protect or fail to control disease. Protection from re-infection relies on the establishment of efficient secondary immune responses that require the generation of antigen-specific \"memory\" B and T lymphocytes.", "Protection from re-infection relies on the establishment of efficient secondary immune responses that require the generation of antigen-specific \"memory\" B and T lymphocytes. The generation and selection of T-cell dependent \"memory\" B cells involves distinct molecular mechanisms: immunoglobulin isotype recombination and somatic hyper mutation, both dependent on the expression of AID [1] . Therefore, a long-standing paradigm defined memory B cells as IgM -IgG + isotype switched cells [2] .", "Therefore, a long-standing paradigm defined memory B cells as IgM -IgG + isotype switched cells [2] . Different lines of evidence indicate that this is not always the case. In humans, it has been shown that some IgM + B cells bear the phenotype of other memory cells, being CD27 + , and carry frequent point mutations in the V region of the Ig genes, suggesting that they must represent highly selected B cell populations [3] .", "In humans, it has been shown that some IgM + B cells bear the phenotype of other memory cells, being CD27 + , and carry frequent point mutations in the V region of the Ig genes, suggesting that they must represent highly selected B cell populations [3] . In mice, populations of CD19 + IgM + able to mount secondary responses have been identified [4] [5] [6] [7] . Overall these findings suggest that the T-cell dependent memory B cell pool comprises distinct subsets of memory B cells with different properties and effector functions [4] [5] [6] .", "Overall these findings suggest that the T-cell dependent memory B cell pool comprises distinct subsets of memory B cells with different properties and effector functions [4] [5] [6] . The biological properties that ensure the long-term persistence of memory and efficient secondary antibody responses have not been yet completely established. While initial studies proposed that after transfer memory B cells faded rapidly [8, 9] suggesting that long-lasting memory required the continuous recruitment of new cells [8] and/or antigen persistence [9, 10] , others suggested that memory B cells were able of extended survival without cell division [11] in the absence of antigen [2] .", "While initial studies proposed that after transfer memory B cells faded rapidly [8, 9] suggesting that long-lasting memory required the continuous recruitment of new cells [8] and/or antigen persistence [9, 10] , others suggested that memory B cells were able of extended survival without cell division [11] in the absence of antigen [2] . Long-term persistence of antibody responses has also been attributed to populations of long-lived plasma cells mainly resident in the bone marrow following immunization [12, 13] . The demonstration of the compartmentalization of \"antibody memory\" into different cellular layers suggested that the separate subsets of memory B cells behave differently.", "The demonstration of the compartmentalization of \"antibody memory\" into different cellular layers suggested that the separate subsets of memory B cells behave differently. Accordingly, it has been reported that IgG + cells that could rapidly respond upon challenge did not persist long, while IgM + cells could generate a second wave of germinal center responses allowing persistence of memory [4] [5] [6] 14] . Currently, immunotherapy approaches using passive antibody transfer [15, 16] ) is limited by the short half-life of immunoglobulin.", "Currently, immunotherapy approaches using passive antibody transfer [15, 16] ) is limited by the short half-life of immunoglobulin. Therefore new therapy strategies may require the adoptive transfer of high-affinity memory B cells, ready to respond and able to persist. The development of these new strategies requires a profound understanding of the mechanisms that regulate memory B cell numbers and ensure long persistence upon adoptive transfer.", "The development of these new strategies requires a profound understanding of the mechanisms that regulate memory B cell numbers and ensure long persistence upon adoptive transfer. Moreover, knowledge of the mechanisms that determine the size of the memory B cell pool may be also critical to device new reconstitution strategies. So far, studies comparing populations of naïve and memory B cells have been hindered both by the vast clonal heterogeneity of the cells involved and by our inability to generate significant numbers of antigen specific memory B cells.", "So far, studies comparing populations of naïve and memory B cells have been hindered both by the vast clonal heterogeneity of the cells involved and by our inability to generate significant numbers of antigen specific memory B cells. Indeed in a normal laboratory mouse the population of B cells bearing a \"memory IgG + phenotype' represent a small fraction of the total B cell pool (<0.5%) and upon immunization the number of the clonal diverse antigen-specific memory B cells generated is generally very limited (<10 3 ) [1, 6] . To circumvent these limits, we decided to compare the properties of homogeneous populations of naïve and memory B cells of known antigen specificity, belonging to the same clone.", "To circumvent these limits, we decided to compare the properties of homogeneous populations of naïve and memory B cells of known antigen specificity, belonging to the same clone. We used SW HEL transgenic mice where B cells bear a high-affinity BCR specific for HEL and are capable of class switch recombination and somatic hypermutation (SHM) [17, 18] . To identify \"memory B cells\" the SW HEL mice were crossed with mice where AID transcription provokes the permanent expression of an YFP reporter in post-germinal center lymphocytes [19] .", "To identify \"memory B cells\" the SW HEL mice were crossed with mice where AID transcription provokes the permanent expression of an YFP reporter in post-germinal center lymphocytes [19] . These mice were in a Rag2-deficient background and therefore contain a pure population of monoclonal HEL-specific B cells. To generate memory cells, purified naïve B cells from the SW HEL .AID/YFP.Rag2 -/mice were transferred into adoptive hosts together with monoclonal OVA-specific CD4 + T cells from OTII.Rag2 -/-TCR transgenic mice.", "To generate memory cells, purified naïve B cells from the SW HEL .AID/YFP.Rag2 -/mice were transferred into adoptive hosts together with monoclonal OVA-specific CD4 + T cells from OTII.Rag2 -/-TCR transgenic mice. Upon immunization with OVA-HEL complexes, we obtained a significant number of persisting HEL-specific IgM + Ig-G -YFP + and IgM -IgG + YFP + memory B cells, number that did not correlate to the number of precursor naïve cells initially injected suggesting that the memory B cell pool is regulated independently. We characterized the functional capacity of these two memory cell types in immune deficient hosts.", "We characterized the functional capacity of these two memory cell types in immune deficient hosts. Mice B6 and B6.Rag2-/- [20] mice were kept at the Centre Des Techniques Avancées (CDTA), Centre National de la Recherche Scientifique (CNRS), Orleans, France; SWHEL.AID/YFP.Rag-/-mice, obtained by crossing SWHEL (18)(a gift of Dr. Robert Brink) and AID/YFP [19] (a gift of Dr. Rafael Casellas) with B6.Rag2-/-mice. OTII.Rag-/-mice were kept in our animal facilities at the Pasteur Institute.", "OTII.Rag-/-mice were kept in our animal facilities at the Pasteur Institute. Experiments were preformed according to Pasteur Institute Safety Committee in accordance with French and European guidelines and the ethics Committee of Paris 1 (permits 2010-0002, -0003 and -0004). Euthanasia of the mice was performed by cervical dislocation.", "Euthanasia of the mice was performed by cervical dislocation. This specific study was approved by the European Research Council (ERC) committee related to the grant AdG09 249740-QSIS. The general status of the mice was controlled daily by monitoring the appearence of obvious pain, distress or suffering (prostration, respiratory issues, loss of weight).", "The general status of the mice was controlled daily by monitoring the appearence of obvious pain, distress or suffering (prostration, respiratory issues, loss of weight). The end-point of the experiment was determined by a loss of more than 20% of the weight or as soon as the distress signs appeared. In this case, experiment was stopped and the animals were euthanized.", "In this case, experiment was stopped and the animals were euthanized. Single-cell suspensions of B cells from spleens and lymph nodes of SW HEL .AID/YFP.Rag -/mice together with CD4 + T cells from spleens and lymph nodes of OTII.Rag -/mice were transferred intravenously into the retro-orbital sinus of B6.Ly5 a IgH a or B6.Rag2 -/recipient mice. Mice received 10 6 HEL + B cells and 10 6 CD4 + T cells unless stated otherwise.", "Mice received 10 6 HEL + B cells and 10 6 CD4 + T cells unless stated otherwise. Mice were immunized 24H later with 1 mg of Ovalbumin coupled to Hen Egg Lysozyme (OVA-HEL) in 50μg of Alu-S-Gel (Serva) we determined as the optimal dose of Ag (data not shown). Naive cells from SW HEL .AID/YFP.Rag -/mice and memory B cells subsets from immunized B6.Rag -/hosts mice were purified from spleens and lymph nodes by flow cytometry sorting.", "Naive cells from SW HEL .AID/YFP.Rag -/mice and memory B cells subsets from immunized B6.Rag -/hosts mice were purified from spleens and lymph nodes by flow cytometry sorting. Single-cell suspensions containing 5×10 4 B cells and 10 6 T cells were transferred intravenously into B6.Rag2 -/recipient hosts. The purity of sorted cells was above 98%.", "The purity of sorted cells was above 98%. 24 h after transfer, mice were immunized with 1 mg of OVA-HEL. Spleen, bone marrow, inguinal and mesenteric lymph nodes single-cell suspensions were stained for cell surface or intracellular proteins with appropriate combinations of the following monoclonal antibodies conjugated to pacific blue, Qdot-655, Brillant Violet 605, allophycocyanin, peridinin chlorophyll protein-cyanine 5.5, phycoerythrin, phycoerythrin-cyanine7: anti-CD19 (6D5), anti-IgM (R6-60.2), anti-IgG1 (X56), anti-CD138 (281-2), anti-Gl7 (Gl7), anti-CD95 (Jo2), anti-CD62L (MEL-14), anti-CD69 (H1-2F3), anti-BAFFR (7H22-E16), anti-CXCR5 (L138D7), anti-IA b (AF6-120.1), anti-CD80 (16-10A1), anti-CD73 (TY-11-8) and anti-PDL2 (TY25) and anti-Ki-67 (mm1) purchased from Becton Dickinson Pharmingen, Biolegend, Invitrogen and eBioscience.", "Spleen, bone marrow, inguinal and mesenteric lymph nodes single-cell suspensions were stained for cell surface or intracellular proteins with appropriate combinations of the following monoclonal antibodies conjugated to pacific blue, Qdot-655, Brillant Violet 605, allophycocyanin, peridinin chlorophyll protein-cyanine 5.5, phycoerythrin, phycoerythrin-cyanine7: anti-CD19 (6D5), anti-IgM (R6-60.2), anti-IgG1 (X56), anti-CD138 (281-2), anti-Gl7 (Gl7), anti-CD95 (Jo2), anti-CD62L (MEL-14), anti-CD69 (H1-2F3), anti-BAFFR (7H22-E16), anti-CXCR5 (L138D7), anti-IA b (AF6-120.1), anti-CD80 (16-10A1), anti-CD73 (TY-11-8) and anti-PDL2 (TY25) and anti-Ki-67 (mm1) purchased from Becton Dickinson Pharmingen, Biolegend, Invitrogen and eBioscience. Cells were also stained with HEL (Sigma) coupled with AF594 using Alexa Fluor1 594 Protein Labeling Kit from Life technologies. Before staining, cells were treated with Fc-Block (CD16/CD32, Becton Dickinson Pharmingen).", "Before staining, cells were treated with Fc-Block (CD16/CD32, Becton Dickinson Pharmingen). Dead cells were excluded during analysis according to their light-scattering characteristics. For intracellular stainings, cells were first stained with antibodies specific for cell surface antigens. Then, cells were fixed and permeabilized according the manufacturer's recommendations (BD Bisciences).", "Then, cells were fixed and permeabilized according the manufacturer's recommendations (BD Bisciences). For proliferation assay, mice were injected i.p. with 50 mg/kg of BrdU (Sigma-Aldrich) and were killed 24 or 72 hours later. Incorporated BrdU was detected intracellularly using anti-BrdU APC-conjugated antibodies according to the manufacturer's recommendations (BD Biosciences).", "Incorporated BrdU was detected intracellularly using anti-BrdU APC-conjugated antibodies according to the manufacturer's recommendations (BD Biosciences). All data acquisitions and analyses were performed with LSRFortessa (Becton Dickinson) interfaced with BD FACSDiva (Becton Dickinson) and FlowJo (Tree Star) software. Subsets of memory B cells were sorted as CD19 + HEL + YFP + IgM + or IgG + and naive cells as CD19 + HEL + YFP -IgM + using a FACSAriaIII flow cytometer.", "Subsets of memory B cells were sorted as CD19 + HEL + YFP + IgM + or IgG + and naive cells as CD19 + HEL + YFP -IgM + using a FACSAriaIII flow cytometer. The purity of the sorted populations varied from 90-95%. Sera HEL-specific Ig concentrations were quantified by ELISA.", "Sera HEL-specific Ig concentrations were quantified by ELISA. Plates were coated with HEL and saturated with PBS-5% Milk. Dilutions of sera were added. After incubation (2 hours, 37˚C) and washing, HRP-labeled anti-mouse IgM or IgG antibodies were added. After incubation and washing, bound antibodies were revealed with the substrate O-phenylenediamine and H2O2.", "After incubation and washing, bound antibodies were revealed with the substrate O-phenylenediamine and H2O2. The reaction was stopped after 10 min. by addition of 10% SDS and the absorbance read at 492nm in a multiscan spectrometer.", "by addition of 10% SDS and the absorbance read at 492nm in a multiscan spectrometer. Ig concentrations were determined by comparing the displacement of the dilution curves in the linear interval between standards at a concentration of 1 mg/ml and the serum samples. The quantification of IgG or IgM secreting cells was assayed by ELISpot technique.", "The quantification of IgG or IgM secreting cells was assayed by ELISpot technique. Briefly, plates were coated with HEL. After saturating, the cells were distributed into the micro wells in RPMI1640-2%FCS. The plates were incubated for 12 h at 37˚C, 5% CO2 atmosphere.", "The plates were incubated for 12 h at 37˚C, 5% CO2 atmosphere. After extensive wash, plates were incubated with goat anti-mouse IgM or anti-IgG labeled with alkaline phosphatase. After washing, the revealing substrate was added (2,3 mM 5-bromo-4-chloro-3-indolyl phosphate diluted in 2-amino-2-methyl-1-proprenolol buffer).", "After washing, the revealing substrate was added (2,3 mM 5-bromo-4-chloro-3-indolyl phosphate diluted in 2-amino-2-methyl-1-proprenolol buffer). Spleens from 14 day-immunized mice were initially fixed with paraformaldehyde and embedded in 4% low-gelling-temperature agarose (type VII-A; Sigma-Aldrich) prepared in PBS. 150μm slices were cut with a vibratome (VT 1000S; Leica) in a bath of ice-cold PBS.", "150μm slices were cut with a vibratome (VT 1000S; Leica) in a bath of ice-cold PBS. For immunolabeling, samples were saturated with PBS supplemented with 10% of fetal calf serum, then were labeled with primary antibodies anti-B220-APC (clone RA3-6B2) and anti-IgD-PE (clone 11-26c.2a) and analyzed with a spinning disk confocal microscope equipped with a CoolSnap HQ2 camera (Photometrics) and a 20x objective. Images were acquired and analyzed with MetaMorph 7 imaging software Molecular Devices).", "Images were acquired and analyzed with MetaMorph 7 imaging software Molecular Devices). Sample means were compared using the Student's t test. Sample means were considered significantly different at p < 0.05.", "Sample means were considered significantly different at p < 0.05. During an immune response the complexity of determinants expressed by immunizing antigen and the degeneracy of antigen-specific recognition results in a vast heterogeneity of responding cells rendering impossible the direct comparison of the properties of naïve and memory B cells belonging to the same clone. We have devised an experimental system that permits the comparison between naïve and memory B cells expressing the same antigen receptor and allows marking permanently memory B cells.", "We have devised an experimental system that permits the comparison between naïve and memory B cells expressing the same antigen receptor and allows marking permanently memory B cells. For that purpose we used SW HEL transgenic mice in a Rag2-deficient background holding a single population of monoclonal B cells, all bearing a high-affinity BCR specific for HEL and capable of class switch recombination and somatic hypermutation (SHM) [17, 18] . To identify antigen-experienced B cells the SW HEL .", "To identify antigen-experienced B cells the SW HEL . Rag2 -/mice were crossed with mice where AID transcription induces the permanent expression of an YFP reporter in post-germinal center lymphocytes [19] . Since in intact Tg mice immune responses were not traceable, probably because of the presence of low level pre-existing anti-HEL antibodies that neutralize the immunizing protein, we used an adoptive cell transfer strategy to study the ability of the high affinity monoclonal B cell to reconstitute response in immune-deficient hosts and generate antibody memory.", "Since in intact Tg mice immune responses were not traceable, probably because of the presence of low level pre-existing anti-HEL antibodies that neutralize the immunizing protein, we used an adoptive cell transfer strategy to study the ability of the high affinity monoclonal B cell to reconstitute response in immune-deficient hosts and generate antibody memory. Purified naïve B cells from the SW HEL .AID/YFP.Rag2 -/mice were transferred into Rag2-deficient mice together with monoclonal OVA-specific CD4 + T helper cells from OTII.Rag2 -/-TCR transgenic mice. The day after, host mice were immunized with OVA-HEL complexes (Fig 1A) .", "The day after, host mice were immunized with OVA-HEL complexes (Fig 1A) . In these conditions, antigenic challenge resulted in B cell activation and the development of significant numbers of CD19 + HEL + AID/YFP + B cells, which were not detected in non-immunized mice or in mice immunized in absence of helper T cells (Fig 1B) . We followed the early kinetics of this response.", "We followed the early kinetics of this response. The number of HEL-specific B cells increased from the initial 2x10 6 transferred to about 15x10 6 at day 14 (Fig 1C left) the B cells expressing AID/YFP being the dominant population (Fig 1C right) . A fraction of the HEL-specific B cells underwent class switch recombination and at day 14 we recovered both IgM + IgG -AID/YFP + and IgM -IgG + AID/YFP + cell populations (Fig 1B) .", "A fraction of the HEL-specific B cells underwent class switch recombination and at day 14 we recovered both IgM + IgG -AID/YFP + and IgM -IgG + AID/YFP + cell populations (Fig 1B) . B cell expansion and phenotypic changes were accompanied by the production of IgM and IgG HEL-specific antibodies ( Fig 1D) . Two weeks after antigenic challenge we observed the formation of germinal centers in the spleen of the host mice ( Fig 1E) .", "Two weeks after antigenic challenge we observed the formation of germinal centers in the spleen of the host mice ( Fig 1E) . Coherently we found that while upon adoptive transfer all B cells expressed CD95, only after antigenic challenge most YFP + B cells expressed the germinal center specific marker GL7 (Fig 1F) . In conclusion, the adoptive cell transfer strategy allowed the development of a primary immune response with B cell activation and expansion, induction of AID expression, class switch recombination, antigen-specific IgM and IgG antibody production and germinal center formation.", "In conclusion, the adoptive cell transfer strategy allowed the development of a primary immune response with B cell activation and expansion, induction of AID expression, class switch recombination, antigen-specific IgM and IgG antibody production and germinal center formation. We studied the evolution of the B cell response. From two weeks onwards the total number of B cells contracted and at four weeks we recovered about 2-4x10 6 cells, number that remained stable up to week 20 (Fig 2A) .", "From two weeks onwards the total number of B cells contracted and at four weeks we recovered about 2-4x10 6 cells, number that remained stable up to week 20 (Fig 2A) . High titers of HEL-specific IgG were kept from week 3 to 8, declined thereafter, but were still significantly elevated 20 weeks later (Fig 2B) . A population of cells secreting HEL-specific Igs was present in the spleen (Fig 2C) , but not in the BM (not shown) even at the late time points.", "A population of cells secreting HEL-specific Igs was present in the spleen (Fig 2C) , but not in the BM (not shown) even at the late time points. About 60% of the recovered cells exhibited the phenotype of antigen-experienced (\"memory\") CD19 + HEL + AID/YFP + expressing either IgM or IgG ( Fig 2D and 2E) . We compared the phenotype of the two AID/YFP + IgM + and AID/YFP + IgM -IgG + memory cell populations recovered with that of the naïve B cells (Fig 2F) .", "We compared the phenotype of the two AID/YFP + IgM + and AID/YFP + IgM -IgG + memory cell populations recovered with that of the naïve B cells (Fig 2F) . We found that antigen-experience and naïve B cells expressed similar levels of CD62L, CD69 and BAFFR (not shown). Antigen-experienced cells presented sustained expression of CD95 and increased levels of PNA, but the vast majority lost expression of the germinal center marker GL7 present at earlier times post-immunization ( Fig 2F compare to Fig 1F) .", "Antigen-experienced cells presented sustained expression of CD95 and increased levels of PNA, but the vast majority lost expression of the germinal center marker GL7 present at earlier times post-immunization ( Fig 2F compare to Fig 1F) . Compared to naïve B cells, AID/ YFP + cells expressed higher levels of CD80 and MHC class II and down-regulated expression of CXCR5 (Fig 2F) . These findings indicate that the post-germinal center AID/YFP + B cells express an activated phenotype [5, 21] , have increased antigen-presenting capacity [22] , but may loose the ability to re-enter primary follicles being CXCR5 low [23] .", "These findings indicate that the post-germinal center AID/YFP + B cells express an activated phenotype [5, 21] , have increased antigen-presenting capacity [22] , but may loose the ability to re-enter primary follicles being CXCR5 low [23] . We have also compared the patterns of gene expression (RNAseq) by naïve, activated (YFPcells of immunized mice) and both populations of YFP + memory cells. The data shows a clear discrimination of naïve and activated/memory cells while indicating only minor differences between both subsets of YFP + memory cells (Fig 3) .", "The data shows a clear discrimination of naïve and activated/memory cells while indicating only minor differences between both subsets of YFP + memory cells (Fig 3) . mRNA was isolated from sort-purified Naïve (CD19 + HEL + YFP -IgM + ) IgM + IgGor IgM -IgG + HEL + CD19 + YFP + memory B cells from spleen of different recipient mice. Total recommended by the manufacturer.", "Total recommended by the manufacturer. The validated libraries were then subjected to DNA sequencing. The analysis is performed using the R software, Bioconductor packages including DESeq2 and the PF2tools package (version 1.2.9) developed at PF2 (Institut Pasteur).", "The analysis is performed using the R software, Bioconductor packages including DESeq2 and the PF2tools package (version 1.2.9) developed at PF2 (Institut Pasteur). Normalization and differential analysis are carried out according to the DESeq2 model and package (version 1.8.1). Fig 3A shows a representative heat map of the different cells populations.", "Fig 3A shows a representative heat map of the different cells populations. Fig 3B shows \n\nLate in the immune response persistent B cell numbers were kept by active cell division as a significant fraction of the cells were Ki67 + (Fig 2G left) and incorporated BrdU (Fig 2G middle) . The frequency of BrdU + cells was higher among the AID/YFPcells (15%) than in the major AID/YFP + memory population (3%) and similar between the IgM + and IgM -AID/ YFP + populations ( Fig 2G middle and not shown) .", "The frequency of BrdU + cells was higher among the AID/YFPcells (15%) than in the major AID/YFP + memory population (3%) and similar between the IgM + and IgM -AID/ YFP + populations ( Fig 2G middle and not shown) . Three days after BrdU pulse populations were clear of BrdU + cells (Fig 2G right) attesting their high division rate. In spite of their increased proliferation rate, memory cells numbers were stable indicating that proliferation may be compensated by cell death as suggested by the frequency of caspase3 + cells (Fig 2H) .", "In spite of their increased proliferation rate, memory cells numbers were stable indicating that proliferation may be compensated by cell death as suggested by the frequency of caspase3 + cells (Fig 2H) . The frequency of Caspase3 + cells was higher among the AID/YFP + cells suggesting that a fraction of these cells may represent cells undergoing terminal differentiation. Importantly, these findings demonstrate that the transfer strategy allowed the generation of significant numbers of persisting antigen-experienced YFP + cells.", "Importantly, these findings demonstrate that the transfer strategy allowed the generation of significant numbers of persisting antigen-experienced YFP + cells. It is not yet known whether the number of antigen-experienced memory B cells correlated to the number of naïve B cells or if it is controlled independently of the initial number of antigenspecific B cells present. To approach this question we transferred different numbers of mature naïve B cells from SW HEL .AID/YFP.Rag2 -/donors (ranging from 10 5 to 5.10 6 ) into Rag2-deficient mice together with an excess of CD4 + T helper cells (10 6 ) and immunize the hosts the day after cell transfer with OVA-HEL in optimal non-limiting quantities.", "To approach this question we transferred different numbers of mature naïve B cells from SW HEL .AID/YFP.Rag2 -/donors (ranging from 10 5 to 5.10 6 ) into Rag2-deficient mice together with an excess of CD4 + T helper cells (10 6 ) and immunize the hosts the day after cell transfer with OVA-HEL in optimal non-limiting quantities. To directly compare the results obtained after the transfer of different all numbers we allowed the responses to reach steady-state eight weeks after antigenic challenge. We studied the amplitude of the immune response by measuring the serum titers of HEL-specific IgG antibodies and enumerating the number of HEL-specific B cells recovered.", "We studied the amplitude of the immune response by measuring the serum titers of HEL-specific IgG antibodies and enumerating the number of HEL-specific B cells recovered. We found that in the presence of excess T cell help, the levels of the HEL-specific IgGs (Fig 4C) , and both the total number of HEL-specific ( Fig 4A) and of memory YFP + B cells recovered (Fig 4B) , did not correlate to the number of antigen specific naïve B cells initially transferred. Memory B cells are defined functionally by their ability to induce secondary IgG antibody responses upon secondary antigenic challenge.", "Memory B cells are defined functionally by their ability to induce secondary IgG antibody responses upon secondary antigenic challenge. We investigated whether the subsets of AID/ YFP + IgM + and AID/YFP + IgM -IgG + antigen-experienced (memory) B cells persisting at late time points could mount secondary IgG responses and persist after secondary transfer. For this purpose we followed two different experimental strategies.", "For this purpose we followed two different experimental strategies. In the first, 5x10 4 cells of either IgM + or IgM -IgG + memory B cells, were transferred with an excess helper OTII CD4 + T cells into secondary Rag-deficient hosts that were boosted with OVA-HEL the day after cell transfer. In the absence of immunization antibody levels were undetectable (not shown) and three weeks after transfer recovery of both memory B cell subsets was about 10-20% of the initial cell input, exceeding naïve B cell recovery (Fig 5A) , supporting the notion that memory B cells may not require specific ligand recognition to survive (2).", "In the absence of immunization antibody levels were undetectable (not shown) and three weeks after transfer recovery of both memory B cell subsets was about 10-20% of the initial cell input, exceeding naïve B cell recovery (Fig 5A) , supporting the notion that memory B cells may not require specific ligand recognition to survive (2). One cannot exclude, however, that cross-reactivity of the BCR transgene with environmental antigens may allow signaling sufficient to maintain naïve and memory cell survival in the absence of HEL [24] . Following immunization, the secondarily transferred AID/YFP + IgM -IgG + cells responded promptly with the exclusive production of significant levels HEL-specific IgG thus confirming their memory statute (11) .", "Following immunization, the secondarily transferred AID/YFP + IgM -IgG + cells responded promptly with the exclusive production of significant levels HEL-specific IgG thus confirming their memory statute (11) . The AID/YFP + IgM + B cells in response to antigenic boost produced only limited amounts of IgM antibodies (Fig 4B) , little IgG antibodies, but did generate GL7 + B cells more efficiently than the IgG + memory B cell population (Fig 5D) . Thus the IgM + subset may contain precursors able to generate a secondary germinal center reaction and a new progeny of IgG + effectors (4).", "Thus the IgM + subset may contain precursors able to generate a secondary germinal center reaction and a new progeny of IgG + effectors (4). With time antibody levels decayed rapidly suggesting that the number of transferred memory B cells declined in the secondary hosts after antigenic boost. Indeed, IgM + and IgG + memory B cells failed to expand and 3 weeks after immunization cell recovery was similar to the retrieval observed in the non-immunized hosts (compare Fig 5E and 5A) .", "Indeed, IgM + and IgG + memory B cells failed to expand and 3 weeks after immunization cell recovery was similar to the retrieval observed in the non-immunized hosts (compare Fig 5E and 5A) . In similar experimental conditions, naïve B cells following immunization expanded, acquired AID/ YFP expression and their numbers more than doubled the number initially injected (Figs 5F and 2A). These data suggest that a significant fraction of the memory B cells generated have a reduced expansion capacity being programmed for rapid differentiation for effector functions.", "These data suggest that a significant fraction of the memory B cells generated have a reduced expansion capacity being programmed for rapid differentiation for effector functions. Besides long-term survival memory B cells must maintain functional activity in the absence of nominal antigen to be fully effective. To test this we used an alternative approach where memory cells were parked in secondary Rag-deficient hosts for 30 days before re-immunization.", "To test this we used an alternative approach where memory cells were parked in secondary Rag-deficient hosts for 30 days before re-immunization. We found that under these conditions antigenic challenge resulted in the production of HEL-specific IgG antibodies and in a 100 fold increase in the number of cells recovered, expansion that largely exceed that observed after immediate challenge (Fig 5G) . The aim of this study was to characterize the fate of activated B cells and the generation of memory B cells.", "The aim of this study was to characterize the fate of activated B cells and the generation of memory B cells. To do this, we adoptively transferred monoclonal B cells into immune deficient hosts followed by immunization in presence of T cell help. This strategy resulted in the development of different B cell memory subsets, namely IgM + and IgG + , as described for in situ generated memory cells [4, 6, 14] .", "This strategy resulted in the development of different B cell memory subsets, namely IgM + and IgG + , as described for in situ generated memory cells [4, 6, 14] . These findings indicate that distinct memory B cell subsets are not the result of the heterogeneity of initially responding naive cells, but originate from the differentiation of a single B cell clone. While studying the respective rate of proliferation of both types of memory B cells, we found the same high rate of proliferation for IgM + and IgG + memory B cells.", "While studying the respective rate of proliferation of both types of memory B cells, we found the same high rate of proliferation for IgM + and IgG + memory B cells. These results contrast with previous published data. First it was been reported that \"in situ\" memory B cells persist as resting non-dividing cells [11, 25] .", "First it was been reported that \"in situ\" memory B cells persist as resting non-dividing cells [11, 25] . However, we have shown that upon adoptive transfer and in absence of competing cells, B cells increase their division rate to occupy the available empty niche [26] , which may explain the higher division rate observed here using this adoptive cell transfer strategy. Secondly, comparing life spans among heterogeneous memory B cell populations it was previously reported a lower division rate among the IgM + subset compared to the IgG + polyclonal subset [6] .", "Secondly, comparing life spans among heterogeneous memory B cell populations it was previously reported a lower division rate among the IgM + subset compared to the IgG + polyclonal subset [6] . Differences in BCR affinity between IgM + and IgG + memory clones may explain the higher division rate previously observed among the IgG + cells [6] . In contrast we compared memory B cell subsets belonging to the same clone bearing the same high affinity BCR.", "In contrast we compared memory B cell subsets belonging to the same clone bearing the same high affinity BCR. Overall these observations support the notion that lymphocyte division rates and life spans are not an intrinsic cell property, but rather determined by the environment and the presence of competing populations [27] . They demonstrate that upon the correct conditions memory B cells can persist by cell division.", "They demonstrate that upon the correct conditions memory B cells can persist by cell division. An important question was whether the number of memory B cells depends on the number of initial naïve B cells. We found that, in the presence of an excess of T cell help, that was not the case.", "We found that, in the presence of an excess of T cell help, that was not the case. However, it was previously reported during polyclonal responses that serum titers of anti-HSA was proportional to the number of cells transferred into irradiated mice [28] . It is possible that limited antigen-specific T-B cell encounters may constraint the number of responding B cells and thus determine linear precursor-progeny between naïve and memory B cells.", "It is possible that limited antigen-specific T-B cell encounters may constraint the number of responding B cells and thus determine linear precursor-progeny between naïve and memory B cells. Our findings indicate that within a single clone the number of precursor naive B cells present in the peripheral B cell pool does determine neither the intensity nor the final number of memory B cells in response to an optimal dose of antigen. They suggest that the size of memory B cell pool may be controlled independently of the number of naïve B cell precursors and that in the absence of clonal competition the memory niche can be filled with a single monoclonal population.", "They suggest that the size of memory B cell pool may be controlled independently of the number of naïve B cell precursors and that in the absence of clonal competition the memory niche can be filled with a single monoclonal population. Considering diverse polyclonal populations, the limited niche for memory cells will imply strong competition among clones resulting in the selection of best fit (high affinity) cells: rare mutated clones being able to out compete more frequent but less avid clones. In our settings, the transgenic memory B cells are likely to counter select any new mutant clones since they express a very high affinity BCR selected in the course of a secondary immune response [29] .", "In our settings, the transgenic memory B cells are likely to counter select any new mutant clones since they express a very high affinity BCR selected in the course of a secondary immune response [29] . Thus, notwithstanding the expression of AID and proliferation we did not detect any BCR VH and VL Ig-chain nucleotide mutations among the recovered memory B cells (not shown). These findings may have implication for vaccination protocols as they indicate that each new antigenic exposure or unrelated immunization would add extra competing clones supporting the need for repeated antigenic boosts to prevent memory B cell attrition.", "These findings may have implication for vaccination protocols as they indicate that each new antigenic exposure or unrelated immunization would add extra competing clones supporting the need for repeated antigenic boosts to prevent memory B cell attrition. They also demonstrate that the memory B cell pool can be reconstituted from a relatively small number of antigen-specific cells. It is likely that the relatively poor memory B cell expansion observed after immediate boost after adoptive transfer could be due to the lack in Rag-deficient hosts of the appropriate environment required for memory B cell survival and function.", "It is likely that the relatively poor memory B cell expansion observed after immediate boost after adoptive transfer could be due to the lack in Rag-deficient hosts of the appropriate environment required for memory B cell survival and function. It should be pointed out that B cell transfer into transgenic ML5 Rag-deficient hosts expressing low levels of HEL [29] resulted in rapid cell loss and recovery suggesting that in these hosts, B cells are trapped by antigen in locations were they are unable to survive (not shown). Nevertheless, it has been shown that B cells can drive the maturation of follicular dendritic cells and the organization of lymphoid follicles [30] .", "Nevertheless, it has been shown that B cells can drive the maturation of follicular dendritic cells and the organization of lymphoid follicles [30] . Similarly, transferred helper cells may also modify their immediate environment. Thus, by allowing lymphocytes to adapt and modify their immediate environment we improved their response and more important, we recovered the memory B cell pool size present in the original donor mice.", "Thus, by allowing lymphocytes to adapt and modify their immediate environment we improved their response and more important, we recovered the memory B cell pool size present in the original donor mice. In this study we show that it is possible to fully reconstitute a primary response and the establishment of antibody memory in immune deficient mice after adoptive transfer of antigen-specific monoclonal B cells together with a population of monoclonal helper T cells. Indeed, it is generally believed that in immune deficiencies, B cell therapy has restricted application due to intrinsic defects of host's lymphoid organs structure that may prevent development of immune responses, germinal center formation, establishment of antibody memory and limit cell survival.", "Indeed, it is generally believed that in immune deficiencies, B cell therapy has restricted application due to intrinsic defects of host's lymphoid organs structure that may prevent development of immune responses, germinal center formation, establishment of antibody memory and limit cell survival. In contrast we showed that after adoptive transfer in immune deficient hosts antigen immunization induced B cell activation and expansion, induction of AID expression, class switch recombination, antigen-specific IgM and IgG antibody production, germinal center formation and the generation of two subsets of AID/YFP + IgM + IgGand AID/YFP + Ig-M -IgG + antigen-experienced B cell subsets able to persist in a lymphopenic environment by cell division mimicking responses obtained in intact non-Tg mice [4] . Upon challenge the AID/YFP + IgM -IgG + cells responded promptly with the production of HEL-specific IgG while the AID/YFP + IgM + B cells secreted only limited amounts of IgM antibodies and fail to produce IgG.", "Upon challenge the AID/YFP + IgM -IgG + cells responded promptly with the production of HEL-specific IgG while the AID/YFP + IgM + B cells secreted only limited amounts of IgM antibodies and fail to produce IgG. In contrast the AID/YFP + IgM + B cells could give rise to new GL7 + B cells, suggesting that full reconstitution of the memory B cell pool may require transfer of the different antigen-experienced B cell subsets. Importantly, we found that the recall responses were more efficient if the transferred memory cells were given the required time to adapt to their new environment, suggesting that a period of accommodation is necessary before the transferred cells are fully capable to respond.", "Importantly, we found that the recall responses were more efficient if the transferred memory cells were given the required time to adapt to their new environment, suggesting that a period of accommodation is necessary before the transferred cells are fully capable to respond. Our findings also show that different processes can modify the survival conditions of memory B cells. Finally, we found that the generation of the memory B cell pool in response to an optimal dose of Ag did not rely on the number of the initially responding B cells, suggesting autonomous homeostatic controls for naïve and memory B cells a property that may allow reconstitution of the memory pool in immune-deficient hosts using a limited number of precursor naïve B cells.", "Finally, we found that the generation of the memory B cell pool in response to an optimal dose of Ag did not rely on the number of the initially responding B cells, suggesting autonomous homeostatic controls for naïve and memory B cells a property that may allow reconstitution of the memory pool in immune-deficient hosts using a limited number of precursor naïve B cells. An autonomous control of the memory B cell pool where each antigenic exposure adds new competing clones supports the notion of vaccination strategies using antigenic boosting to prevent memory B cell attrition. Overall the findings reported demonstrate that it is possible to reconstitute the memory B cell pool of an immune deficient host with an artificially induced population of monoclonal high affinity memory B cells." ]

[ "nan\n\nText: The main challenge may include (1) early identification of outbreak, (2) rapid expansion of patients, (3) high risk of nosocomial transmission, (4) unpredictability of size impacted, and (5) lack of backup resource. These challenges have caused severe shortage of healthcare workers, medical materials, and beds with isolation. The Spring Festival holiday has greatly aggravated the shortage of human resources and heavy traffic flow due to the vacation of healthy workers and factory workers, which further magnified the risk of transmission.", "The Spring Festival holiday has greatly aggravated the shortage of human resources and heavy traffic flow due to the vacation of healthy workers and factory workers, which further magnified the risk of transmission. The key point is to discriminate the infectious disease outbreak from regular clustering cases of flu-like diseases at early stage. There is a trade-off between false alarm causing population panic and delayed identification leading to social crisis.", "There is a trade-off between false alarm causing population panic and delayed identification leading to social crisis. Early identification of 2019-nCoV infection presents a major challenge for the frontline clinicians. Its clinical symptoms largely overlap with those of common acute respiratory illnesses, including fever (98%), cough (76%), and diarrhea (3%), often more severe in older adults with pre-existing chronic comorbidities [1] .", "Its clinical symptoms largely overlap with those of common acute respiratory illnesses, including fever (98%), cough (76%), and diarrhea (3%), often more severe in older adults with pre-existing chronic comorbidities [1] . Usually, the laboratory abnormalities include lymphocytopenia and hypoxemia [1] . The initial chest radiographs may vary from minimal abnormality to bilateral ground-glass opacity or subsegmental areas of consolidation [1] .", "The initial chest radiographs may vary from minimal abnormality to bilateral ground-glass opacity or subsegmental areas of consolidation [1] . In addition, asymptomatic cases and lack of diagnosis kits result in delayed or even missed diagnosis inevitable and makes many other patients, visitors, and healthcare workers exposed to the 2019-nCoV infection. Critical care response to the outbreak of coronavirus should happen not only at the level of hospital, but also at the level of the city which is dominated by the government.", "Critical care response to the outbreak of coronavirus should happen not only at the level of hospital, but also at the level of the city which is dominated by the government. At the early stage, the size of the patients' population is not beyond the capability of local infectious diseases hospital (IDH). The general hospital is responsible for fever triage, identifying suspected cases, and transferring to the local IDH.", "The general hospital is responsible for fever triage, identifying suspected cases, and transferring to the local IDH. Such a plan is mandatory for every hospital. Shenzhen city has established a preexisting Infectious Disease Epidemic Plan (IDEP), which has facilitated managing and containing local outbreak of the 2019-nCoV.", "Shenzhen city has established a preexisting Infectious Disease Epidemic Plan (IDEP), which has facilitated managing and containing local outbreak of the 2019-nCoV. In case the patient load exceeds the hospital capability of the IDH, new IDHs should be considered either by building a temporary new IDH or reconstructing an existing hospital. Wuhan, the epicenter of the outbreak, is racing against time to build two specialized hospitals for nCoV patients, namely Huoshenshan and Leishenshan hospital, whereas a different strategy has been undertaken in Shenzhen city by reconstructing an existing hospital to become an IDH with capability of 800 beds.", "Wuhan, the epicenter of the outbreak, is racing against time to build two specialized hospitals for nCoV patients, namely Huoshenshan and Leishenshan hospital, whereas a different strategy has been undertaken in Shenzhen city by reconstructing an existing hospital to become an IDH with capability of 800 beds. 2019-nCoV patients should be admitted to singlebedded, negative pressure rooms in isolated units with intensive care and monitoring [2] . Clinical engineering should have plans to reconstruct standard rooms [2] .", "Clinical engineering should have plans to reconstruct standard rooms [2] . Retrofitting the rooms with externally exhausted HEPA filters may be an expedient solution. Also, the general hospital may consider procedures such as suspending elective surgeries, canceling ambulatory clinics and outpatient diagnostic procedures, transferring patients to other institutions, and restricting hospital visitors [2] .", "Also, the general hospital may consider procedures such as suspending elective surgeries, canceling ambulatory clinics and outpatient diagnostic procedures, transferring patients to other institutions, and restricting hospital visitors [2] . More importantly, because the hospitals' ability to respond to the outbreak largely depends on their available ICU beds, the plan to increase ICU bed capacity needs to be determined. Caring for 2019-nCoV patients represents a substantial exposure risk for ICU staff because of the following reasons: highly contagious with multiple transmission route, high exposure dose, long daily contact hours, and ICU stay.", "Caring for 2019-nCoV patients represents a substantial exposure risk for ICU staff because of the following reasons: highly contagious with multiple transmission route, high exposure dose, long daily contact hours, and ICU stay. The basic reproductive number was estimated to be 2.2 (95% CI, 1.4 to 3.9) [3] , or as high as between 3.6 and 4.0 [4] . The 2019-nCoV is proved to be transmitted by respiratory droplets, contact, and fecal-oral, even transmission through the eye is possible [5, 6] .", "The 2019-nCoV is proved to be transmitted by respiratory droplets, contact, and fecal-oral, even transmission through the eye is possible [5, 6] . The higher viral load and aerosol-generating procedures, such as noninvasive ventilation, magnify the exposure and transmission risk [2, 7, 8] . Moreover, virus shedding can be prolonged and last for > 3 weeks according to some literature and our unpublished data [2] .", "Moreover, virus shedding can be prolonged and last for > 3 weeks according to some literature and our unpublished data [2] . Healthcare providers and those in contact with infected patients should utilize contact, droplet, and airborne precautions with N95 respirator. Strict infection prevention and control practices have been implemented and audited in our units following the infection prevention and control plan published by China's National Health Committee (CNHC).", "Strict infection prevention and control practices have been implemented and audited in our units following the infection prevention and control plan published by China's National Health Committee (CNHC). In addition, wellequipped fever clinic as triage station with trained staff knowing 2019-nCoV case definitions is established. For suspected 2019-nCoV infection, several key points are crucial procedures: recording a detailed history, standardizing pneumonia workup, obtaining lower respiratory tract specimens [2, 8] , and implementing droplet isolation to break the transmission chain in the healthcare setting [2] .", "For suspected 2019-nCoV infection, several key points are crucial procedures: recording a detailed history, standardizing pneumonia workup, obtaining lower respiratory tract specimens [2, 8] , and implementing droplet isolation to break the transmission chain in the healthcare setting [2] . The risk of 2019-nCoV exposure may cause significant psychosocial stress on healthcare workers [2] . The death of a retired ENT physician from a 2019-nCoV infection has added to fears in January 2020.", "The death of a retired ENT physician from a 2019-nCoV infection has added to fears in January 2020. Psychotherapists have also been invited to join medical teams to evaluate and deal with potential stress and depression for the safety of the healthcare workers. Critical management 2019-nCoV management was largely supportive, including intubation, early prone positioning, neuromuscular blockade, and extracorporeal membrane oxygenation (ECMO) according to the recommendations updated by CNHC.", "Critical management 2019-nCoV management was largely supportive, including intubation, early prone positioning, neuromuscular blockade, and extracorporeal membrane oxygenation (ECMO) according to the recommendations updated by CNHC. Low-dose systematic corticosteroids, lopinavir/ritonavir, and atomization inhalation of interferon were encouraged. These critical managements have worked well so far, as our 2019-nCoV patients had zero mortality.", "These critical managements have worked well so far, as our 2019-nCoV patients had zero mortality. On the contrary, the previously reported mortality of 2019-nCoV patients in Wuhan ranged from 11 to 15% [1, 9] ." ]

[ "nan\n\nText: The main challenge may include (1) early identification of outbreak, (2) rapid expansion of patients, (3) high risk of nosocomial transmission, (4) unpredictability of size impacted, and (5) lack of backup resource. These challenges have caused severe shortage of healthcare workers, medical materials, and beds with isolation. The Spring Festival holiday has greatly aggravated the shortage of human resources and heavy traffic flow due to the vacation of healthy workers and factory workers, which further magnified the risk of transmission.", "The Spring Festival holiday has greatly aggravated the shortage of human resources and heavy traffic flow due to the vacation of healthy workers and factory workers, which further magnified the risk of transmission. The key point is to discriminate the infectious disease outbreak from regular clustering cases of flu-like diseases at early stage. There is a trade-off between false alarm causing population panic and delayed identification leading to social crisis.", "There is a trade-off between false alarm causing population panic and delayed identification leading to social crisis. Early identification of 2019-nCoV infection presents a major challenge for the frontline clinicians. Its clinical symptoms largely overlap with those of common acute respiratory illnesses, including fever (98%), cough (76%), and diarrhea (3%), often more severe in older adults with pre-existing chronic comorbidities [1] .", "Its clinical symptoms largely overlap with those of common acute respiratory illnesses, including fever (98%), cough (76%), and diarrhea (3%), often more severe in older adults with pre-existing chronic comorbidities [1] . Usually, the laboratory abnormalities include lymphocytopenia and hypoxemia [1] . The initial chest radiographs may vary from minimal abnormality to bilateral ground-glass opacity or subsegmental areas of consolidation [1] .", "The initial chest radiographs may vary from minimal abnormality to bilateral ground-glass opacity or subsegmental areas of consolidation [1] . In addition, asymptomatic cases and lack of diagnosis kits result in delayed or even missed diagnosis inevitable and makes many other patients, visitors, and healthcare workers exposed to the 2019-nCoV infection. Critical care response to the outbreak of coronavirus should happen not only at the level of hospital, but also at the level of the city which is dominated by the government.", "Critical care response to the outbreak of coronavirus should happen not only at the level of hospital, but also at the level of the city which is dominated by the government. At the early stage, the size of the patients' population is not beyond the capability of local infectious diseases hospital (IDH). The general hospital is responsible for fever triage, identifying suspected cases, and transferring to the local IDH.", "The general hospital is responsible for fever triage, identifying suspected cases, and transferring to the local IDH. Such a plan is mandatory for every hospital. Shenzhen city has established a preexisting Infectious Disease Epidemic Plan (IDEP), which has facilitated managing and containing local outbreak of the 2019-nCoV.", "Shenzhen city has established a preexisting Infectious Disease Epidemic Plan (IDEP), which has facilitated managing and containing local outbreak of the 2019-nCoV. In case the patient load exceeds the hospital capability of the IDH, new IDHs should be considered either by building a temporary new IDH or reconstructing an existing hospital. Wuhan, the epicenter of the outbreak, is racing against time to build two specialized hospitals for nCoV patients, namely Huoshenshan and Leishenshan hospital, whereas a different strategy has been undertaken in Shenzhen city by reconstructing an existing hospital to become an IDH with capability of 800 beds.", "Wuhan, the epicenter of the outbreak, is racing against time to build two specialized hospitals for nCoV patients, namely Huoshenshan and Leishenshan hospital, whereas a different strategy has been undertaken in Shenzhen city by reconstructing an existing hospital to become an IDH with capability of 800 beds. 2019-nCoV patients should be admitted to singlebedded, negative pressure rooms in isolated units with intensive care and monitoring [2] . Clinical engineering should have plans to reconstruct standard rooms [2] .", "Clinical engineering should have plans to reconstruct standard rooms [2] . Retrofitting the rooms with externally exhausted HEPA filters may be an expedient solution. Also, the general hospital may consider procedures such as suspending elective surgeries, canceling ambulatory clinics and outpatient diagnostic procedures, transferring patients to other institutions, and restricting hospital visitors [2] .", "Also, the general hospital may consider procedures such as suspending elective surgeries, canceling ambulatory clinics and outpatient diagnostic procedures, transferring patients to other institutions, and restricting hospital visitors [2] . More importantly, because the hospitals' ability to respond to the outbreak largely depends on their available ICU beds, the plan to increase ICU bed capacity needs to be determined. Caring for 2019-nCoV patients represents a substantial exposure risk for ICU staff because of the following reasons: highly contagious with multiple transmission route, high exposure dose, long daily contact hours, and ICU stay.", "Caring for 2019-nCoV patients represents a substantial exposure risk for ICU staff because of the following reasons: highly contagious with multiple transmission route, high exposure dose, long daily contact hours, and ICU stay. The basic reproductive number was estimated to be 2.2 (95% CI, 1.4 to 3.9) [3] , or as high as between 3.6 and 4.0 [4] . The 2019-nCoV is proved to be transmitted by respiratory droplets, contact, and fecal-oral, even transmission through the eye is possible [5, 6] .", "The 2019-nCoV is proved to be transmitted by respiratory droplets, contact, and fecal-oral, even transmission through the eye is possible [5, 6] . The higher viral load and aerosol-generating procedures, such as noninvasive ventilation, magnify the exposure and transmission risk [2, 7, 8] . Moreover, virus shedding can be prolonged and last for > 3 weeks according to some literature and our unpublished data [2] .", "Moreover, virus shedding can be prolonged and last for > 3 weeks according to some literature and our unpublished data [2] . Healthcare providers and those in contact with infected patients should utilize contact, droplet, and airborne precautions with N95 respirator. Strict infection prevention and control practices have been implemented and audited in our units following the infection prevention and control plan published by China's National Health Committee (CNHC).", "Strict infection prevention and control practices have been implemented and audited in our units following the infection prevention and control plan published by China's National Health Committee (CNHC). In addition, wellequipped fever clinic as triage station with trained staff knowing 2019-nCoV case definitions is established. For suspected 2019-nCoV infection, several key points are crucial procedures: recording a detailed history, standardizing pneumonia workup, obtaining lower respiratory tract specimens [2, 8] , and implementing droplet isolation to break the transmission chain in the healthcare setting [2] .", "For suspected 2019-nCoV infection, several key points are crucial procedures: recording a detailed history, standardizing pneumonia workup, obtaining lower respiratory tract specimens [2, 8] , and implementing droplet isolation to break the transmission chain in the healthcare setting [2] . The risk of 2019-nCoV exposure may cause significant psychosocial stress on healthcare workers [2] . The death of a retired ENT physician from a 2019-nCoV infection has added to fears in January 2020.", "The death of a retired ENT physician from a 2019-nCoV infection has added to fears in January 2020. Psychotherapists have also been invited to join medical teams to evaluate and deal with potential stress and depression for the safety of the healthcare workers. Critical management 2019-nCoV management was largely supportive, including intubation, early prone positioning, neuromuscular blockade, and extracorporeal membrane oxygenation (ECMO) according to the recommendations updated by CNHC.", "Critical management 2019-nCoV management was largely supportive, including intubation, early prone positioning, neuromuscular blockade, and extracorporeal membrane oxygenation (ECMO) according to the recommendations updated by CNHC. Low-dose systematic corticosteroids, lopinavir/ritonavir, and atomization inhalation of interferon were encouraged. These critical managements have worked well so far, as our 2019-nCoV patients had zero mortality.", "These critical managements have worked well so far, as our 2019-nCoV patients had zero mortality. On the contrary, the previously reported mortality of 2019-nCoV patients in Wuhan ranged from 11 to 15% [1, 9] ." ]

[ "nan\n\nText: The main challenge may include (1) early identification of outbreak, (2) rapid expansion of patients, (3) high risk of nosocomial transmission, (4) unpredictability of size impacted, and (5) lack of backup resource. These challenges have caused severe shortage of healthcare workers, medical materials, and beds with isolation. The Spring Festival holiday has greatly aggravated the shortage of human resources and heavy traffic flow due to the vacation of healthy workers and factory workers, which further magnified the risk of transmission.", "The Spring Festival holiday has greatly aggravated the shortage of human resources and heavy traffic flow due to the vacation of healthy workers and factory workers, which further magnified the risk of transmission. The key point is to discriminate the infectious disease outbreak from regular clustering cases of flu-like diseases at early stage. There is a trade-off between false alarm causing population panic and delayed identification leading to social crisis.", "There is a trade-off between false alarm causing population panic and delayed identification leading to social crisis. Early identification of 2019-nCoV infection presents a major challenge for the frontline clinicians. Its clinical symptoms largely overlap with those of common acute respiratory illnesses, including fever (98%), cough (76%), and diarrhea (3%), often more severe in older adults with pre-existing chronic comorbidities [1] .", "Its clinical symptoms largely overlap with those of common acute respiratory illnesses, including fever (98%), cough (76%), and diarrhea (3%), often more severe in older adults with pre-existing chronic comorbidities [1] . Usually, the laboratory abnormalities include lymphocytopenia and hypoxemia [1] . The initial chest radiographs may vary from minimal abnormality to bilateral ground-glass opacity or subsegmental areas of consolidation [1] .", "The initial chest radiographs may vary from minimal abnormality to bilateral ground-glass opacity or subsegmental areas of consolidation [1] . In addition, asymptomatic cases and lack of diagnosis kits result in delayed or even missed diagnosis inevitable and makes many other patients, visitors, and healthcare workers exposed to the 2019-nCoV infection. Critical care response to the outbreak of coronavirus should happen not only at the level of hospital, but also at the level of the city which is dominated by the government.", "Critical care response to the outbreak of coronavirus should happen not only at the level of hospital, but also at the level of the city which is dominated by the government. At the early stage, the size of the patients' population is not beyond the capability of local infectious diseases hospital (IDH). The general hospital is responsible for fever triage, identifying suspected cases, and transferring to the local IDH.", "The general hospital is responsible for fever triage, identifying suspected cases, and transferring to the local IDH. Such a plan is mandatory for every hospital. Shenzhen city has established a preexisting Infectious Disease Epidemic Plan (IDEP), which has facilitated managing and containing local outbreak of the 2019-nCoV.", "Shenzhen city has established a preexisting Infectious Disease Epidemic Plan (IDEP), which has facilitated managing and containing local outbreak of the 2019-nCoV. In case the patient load exceeds the hospital capability of the IDH, new IDHs should be considered either by building a temporary new IDH or reconstructing an existing hospital. Wuhan, the epicenter of the outbreak, is racing against time to build two specialized hospitals for nCoV patients, namely Huoshenshan and Leishenshan hospital, whereas a different strategy has been undertaken in Shenzhen city by reconstructing an existing hospital to become an IDH with capability of 800 beds.", "Wuhan, the epicenter of the outbreak, is racing against time to build two specialized hospitals for nCoV patients, namely Huoshenshan and Leishenshan hospital, whereas a different strategy has been undertaken in Shenzhen city by reconstructing an existing hospital to become an IDH with capability of 800 beds. 2019-nCoV patients should be admitted to singlebedded, negative pressure rooms in isolated units with intensive care and monitoring [2] . Clinical engineering should have plans to reconstruct standard rooms [2] .", "Clinical engineering should have plans to reconstruct standard rooms [2] . Retrofitting the rooms with externally exhausted HEPA filters may be an expedient solution. Also, the general hospital may consider procedures such as suspending elective surgeries, canceling ambulatory clinics and outpatient diagnostic procedures, transferring patients to other institutions, and restricting hospital visitors [2] .", "Also, the general hospital may consider procedures such as suspending elective surgeries, canceling ambulatory clinics and outpatient diagnostic procedures, transferring patients to other institutions, and restricting hospital visitors [2] . More importantly, because the hospitals' ability to respond to the outbreak largely depends on their available ICU beds, the plan to increase ICU bed capacity needs to be determined. Caring for 2019-nCoV patients represents a substantial exposure risk for ICU staff because of the following reasons: highly contagious with multiple transmission route, high exposure dose, long daily contact hours, and ICU stay.", "Caring for 2019-nCoV patients represents a substantial exposure risk for ICU staff because of the following reasons: highly contagious with multiple transmission route, high exposure dose, long daily contact hours, and ICU stay. The basic reproductive number was estimated to be 2.2 (95% CI, 1.4 to 3.9) [3] , or as high as between 3.6 and 4.0 [4] . The 2019-nCoV is proved to be transmitted by respiratory droplets, contact, and fecal-oral, even transmission through the eye is possible [5, 6] .", "The 2019-nCoV is proved to be transmitted by respiratory droplets, contact, and fecal-oral, even transmission through the eye is possible [5, 6] . The higher viral load and aerosol-generating procedures, such as noninvasive ventilation, magnify the exposure and transmission risk [2, 7, 8] . Moreover, virus shedding can be prolonged and last for > 3 weeks according to some literature and our unpublished data [2] .", "Moreover, virus shedding can be prolonged and last for > 3 weeks according to some literature and our unpublished data [2] . Healthcare providers and those in contact with infected patients should utilize contact, droplet, and airborne precautions with N95 respirator. Strict infection prevention and control practices have been implemented and audited in our units following the infection prevention and control plan published by China's National Health Committee (CNHC).", "Strict infection prevention and control practices have been implemented and audited in our units following the infection prevention and control plan published by China's National Health Committee (CNHC). In addition, wellequipped fever clinic as triage station with trained staff knowing 2019-nCoV case definitions is established. For suspected 2019-nCoV infection, several key points are crucial procedures: recording a detailed history, standardizing pneumonia workup, obtaining lower respiratory tract specimens [2, 8] , and implementing droplet isolation to break the transmission chain in the healthcare setting [2] .", "For suspected 2019-nCoV infection, several key points are crucial procedures: recording a detailed history, standardizing pneumonia workup, obtaining lower respiratory tract specimens [2, 8] , and implementing droplet isolation to break the transmission chain in the healthcare setting [2] . The risk of 2019-nCoV exposure may cause significant psychosocial stress on healthcare workers [2] . The death of a retired ENT physician from a 2019-nCoV infection has added to fears in January 2020.", "The death of a retired ENT physician from a 2019-nCoV infection has added to fears in January 2020. Psychotherapists have also been invited to join medical teams to evaluate and deal with potential stress and depression for the safety of the healthcare workers. Critical management 2019-nCoV management was largely supportive, including intubation, early prone positioning, neuromuscular blockade, and extracorporeal membrane oxygenation (ECMO) according to the recommendations updated by CNHC.", "Critical management 2019-nCoV management was largely supportive, including intubation, early prone positioning, neuromuscular blockade, and extracorporeal membrane oxygenation (ECMO) according to the recommendations updated by CNHC. Low-dose systematic corticosteroids, lopinavir/ritonavir, and atomization inhalation of interferon were encouraged. These critical managements have worked well so far, as our 2019-nCoV patients had zero mortality.", "These critical managements have worked well so far, as our 2019-nCoV patients had zero mortality. On the contrary, the previously reported mortality of 2019-nCoV patients in Wuhan ranged from 11 to 15% [1, 9] ." ]

[ "nan\n\nText: The main challenge may include (1) early identification of outbreak, (2) rapid expansion of patients, (3) high risk of nosocomial transmission, (4) unpredictability of size impacted, and (5) lack of backup resource. These challenges have caused severe shortage of healthcare workers, medical materials, and beds with isolation. The Spring Festival holiday has greatly aggravated the shortage of human resources and heavy traffic flow due to the vacation of healthy workers and factory workers, which further magnified the risk of transmission.", "The Spring Festival holiday has greatly aggravated the shortage of human resources and heavy traffic flow due to the vacation of healthy workers and factory workers, which further magnified the risk of transmission. The key point is to discriminate the infectious disease outbreak from regular clustering cases of flu-like diseases at early stage. There is a trade-off between false alarm causing population panic and delayed identification leading to social crisis.", "There is a trade-off between false alarm causing population panic and delayed identification leading to social crisis. Early identification of 2019-nCoV infection presents a major challenge for the frontline clinicians. Its clinical symptoms largely overlap with those of common acute respiratory illnesses, including fever (98%), cough (76%), and diarrhea (3%), often more severe in older adults with pre-existing chronic comorbidities [1] .", "Its clinical symptoms largely overlap with those of common acute respiratory illnesses, including fever (98%), cough (76%), and diarrhea (3%), often more severe in older adults with pre-existing chronic comorbidities [1] . Usually, the laboratory abnormalities include lymphocytopenia and hypoxemia [1] . The initial chest radiographs may vary from minimal abnormality to bilateral ground-glass opacity or subsegmental areas of consolidation [1] .", "The initial chest radiographs may vary from minimal abnormality to bilateral ground-glass opacity or subsegmental areas of consolidation [1] . In addition, asymptomatic cases and lack of diagnosis kits result in delayed or even missed diagnosis inevitable and makes many other patients, visitors, and healthcare workers exposed to the 2019-nCoV infection. Critical care response to the outbreak of coronavirus should happen not only at the level of hospital, but also at the level of the city which is dominated by the government.", "Critical care response to the outbreak of coronavirus should happen not only at the level of hospital, but also at the level of the city which is dominated by the government. At the early stage, the size of the patients' population is not beyond the capability of local infectious diseases hospital (IDH). The general hospital is responsible for fever triage, identifying suspected cases, and transferring to the local IDH.", "The general hospital is responsible for fever triage, identifying suspected cases, and transferring to the local IDH. Such a plan is mandatory for every hospital. Shenzhen city has established a preexisting Infectious Disease Epidemic Plan (IDEP), which has facilitated managing and containing local outbreak of the 2019-nCoV.", "Shenzhen city has established a preexisting Infectious Disease Epidemic Plan (IDEP), which has facilitated managing and containing local outbreak of the 2019-nCoV. In case the patient load exceeds the hospital capability of the IDH, new IDHs should be considered either by building a temporary new IDH or reconstructing an existing hospital. Wuhan, the epicenter of the outbreak, is racing against time to build two specialized hospitals for nCoV patients, namely Huoshenshan and Leishenshan hospital, whereas a different strategy has been undertaken in Shenzhen city by reconstructing an existing hospital to become an IDH with capability of 800 beds.", "Wuhan, the epicenter of the outbreak, is racing against time to build two specialized hospitals for nCoV patients, namely Huoshenshan and Leishenshan hospital, whereas a different strategy has been undertaken in Shenzhen city by reconstructing an existing hospital to become an IDH with capability of 800 beds. 2019-nCoV patients should be admitted to singlebedded, negative pressure rooms in isolated units with intensive care and monitoring [2] . Clinical engineering should have plans to reconstruct standard rooms [2] .", "Clinical engineering should have plans to reconstruct standard rooms [2] . Retrofitting the rooms with externally exhausted HEPA filters may be an expedient solution. Also, the general hospital may consider procedures such as suspending elective surgeries, canceling ambulatory clinics and outpatient diagnostic procedures, transferring patients to other institutions, and restricting hospital visitors [2] .", "Also, the general hospital may consider procedures such as suspending elective surgeries, canceling ambulatory clinics and outpatient diagnostic procedures, transferring patients to other institutions, and restricting hospital visitors [2] . More importantly, because the hospitals' ability to respond to the outbreak largely depends on their available ICU beds, the plan to increase ICU bed capacity needs to be determined. Caring for 2019-nCoV patients represents a substantial exposure risk for ICU staff because of the following reasons: highly contagious with multiple transmission route, high exposure dose, long daily contact hours, and ICU stay.", "Caring for 2019-nCoV patients represents a substantial exposure risk for ICU staff because of the following reasons: highly contagious with multiple transmission route, high exposure dose, long daily contact hours, and ICU stay. The basic reproductive number was estimated to be 2.2 (95% CI, 1.4 to 3.9) [3] , or as high as between 3.6 and 4.0 [4] . The 2019-nCoV is proved to be transmitted by respiratory droplets, contact, and fecal-oral, even transmission through the eye is possible [5, 6] .", "The 2019-nCoV is proved to be transmitted by respiratory droplets, contact, and fecal-oral, even transmission through the eye is possible [5, 6] . The higher viral load and aerosol-generating procedures, such as noninvasive ventilation, magnify the exposure and transmission risk [2, 7, 8] . Moreover, virus shedding can be prolonged and last for > 3 weeks according to some literature and our unpublished data [2] .", "Moreover, virus shedding can be prolonged and last for > 3 weeks according to some literature and our unpublished data [2] . Healthcare providers and those in contact with infected patients should utilize contact, droplet, and airborne precautions with N95 respirator. Strict infection prevention and control practices have been implemented and audited in our units following the infection prevention and control plan published by China's National Health Committee (CNHC).", "Strict infection prevention and control practices have been implemented and audited in our units following the infection prevention and control plan published by China's National Health Committee (CNHC). In addition, wellequipped fever clinic as triage station with trained staff knowing 2019-nCoV case definitions is established. For suspected 2019-nCoV infection, several key points are crucial procedures: recording a detailed history, standardizing pneumonia workup, obtaining lower respiratory tract specimens [2, 8] , and implementing droplet isolation to break the transmission chain in the healthcare setting [2] .", "For suspected 2019-nCoV infection, several key points are crucial procedures: recording a detailed history, standardizing pneumonia workup, obtaining lower respiratory tract specimens [2, 8] , and implementing droplet isolation to break the transmission chain in the healthcare setting [2] . The risk of 2019-nCoV exposure may cause significant psychosocial stress on healthcare workers [2] . The death of a retired ENT physician from a 2019-nCoV infection has added to fears in January 2020.", "The death of a retired ENT physician from a 2019-nCoV infection has added to fears in January 2020. Psychotherapists have also been invited to join medical teams to evaluate and deal with potential stress and depression for the safety of the healthcare workers. Critical management 2019-nCoV management was largely supportive, including intubation, early prone positioning, neuromuscular blockade, and extracorporeal membrane oxygenation (ECMO) according to the recommendations updated by CNHC.", "Critical management 2019-nCoV management was largely supportive, including intubation, early prone positioning, neuromuscular blockade, and extracorporeal membrane oxygenation (ECMO) according to the recommendations updated by CNHC. Low-dose systematic corticosteroids, lopinavir/ritonavir, and atomization inhalation of interferon were encouraged. These critical managements have worked well so far, as our 2019-nCoV patients had zero mortality.", "These critical managements have worked well so far, as our 2019-nCoV patients had zero mortality. On the contrary, the previously reported mortality of 2019-nCoV patients in Wuhan ranged from 11 to 15% [1, 9] ." ]

[ "Information about the open-access article 'Statistics-Based Predictions of Coronavirus Epidemic Spreading in Mainland China' in DOAJ. DOAJ is an online directory that indexes and provides access to quality open access, peer-reviewed journals. Text: Here, we consider the development of an epidemic outbreak caused by coronavirus COVID-19 (the previous name was 2019-nCoV) (see e.g., [1] [2] [3] ).", "Text: Here, we consider the development of an epidemic outbreak caused by coronavirus COVID-19 (the previous name was 2019-nCoV) (see e.g., [1] [2] [3] ). Since long-term data are available only for mainland China, we will try to predict the number of coronavirus victims V (number of persons who caught the infection and got sick) only in this area. The first estimations of V(t) exponential growth versus time t, typical for the initial stages of every epidemic (see e.g., [4] ) have been done in [3] .", "The first estimations of V(t) exponential growth versus time t, typical for the initial stages of every epidemic (see e.g., [4] ) have been done in [3] . For long-time predictions, more complicated mathematical models are necessary. For example, a susceptible-exposed-infectious-recovered (SEIR) model was used in [2] .", "For example, a susceptible-exposed-infectious-recovered (SEIR) model was used in [2] . Nevertheless, complicated models need more effort for unknown parameters identification. This procedure may be especially difficult if reliable data are limited.", "This procedure may be especially difficult if reliable data are limited. In this study, we use the known SIR model for the dynamics of an epidemic [4] [5] [6] [7] [8] . For the parameter identification, we will use the exact solution of the SIR set of linear equations and statistical approach developed in [4] (tested also in [9] ).", "For the parameter identification, we will use the exact solution of the SIR set of linear equations and statistical approach developed in [4] (tested also in [9] ). These methods were applied for investigation of the children disease, which occurred in Chernivtsi (Ukraine) in 1988-1989. We will estimate some of the epidemic characteristics and present the dependencies for victim numbers, infected and removed persons versus time.", "We will estimate some of the epidemic characteristics and present the dependencies for victim numbers, infected and removed persons versus time. We shall analyze the daily data for the number of confirmed cases in mainland China, which origins from the National Health Commission of the People's Republic of China [1] . A part of the official diagram (its version, presented on February 15, 2020) is shown in Fig.", "A part of the official diagram (its version, presented on February 15, 2020) is shown in Fig. 1 . For calculations, we have used the data for the period of time from January 16 to February 9, 2020. The numbers shown after February 9 were used for verification of predictions.", "The numbers shown after February 9 were used for verification of predictions. On February 12, 2020, the National Health Commission of the People's Republic of China has added 12289 new cases (not previously included in official counts) as \"clinically diagnosed cases\". The cases, reported by this official organization before, have the name of \"tested confirmed cases\" [1] .", "The cases, reported by this official organization before, have the name of \"tested confirmed cases\" [1] . To avoid confusiong, we will denote \"tested confirmed cases\" as Wj; j corresponds to the different time moments tj (see the Table) . Let us denote the \"clinically diagnosed cases\" as Qj.", "Let us denote the \"clinically diagnosed cases\" as Qj. The sum of Wj and Qj is shown in the last column in Fig. 1 and in the Table. The Table shows that the precise time of the epidemic beginning t0 is unknown.", "The Table shows that the precise time of the epidemic beginning t0 is unknown. Therefore, the optimization procedures have to determine the optimal value of this parameter as well as for other parameters of SIR model. The sum of \"tested confirmed cases\" and \"clinically diagnosed cases\" Wj + Qj 16 0 45 1 16 14380 Unknown 17 1 62 2 17 17205 Unknown 18 2 121 3 18 20440 Unknown 19 3 198 4 19 24324 Unknown 20 4 291 5 20 28018 Unknown 21 5 440 6 21 31161 Unknown 22 6 571 7 22 34568 Unknown 23 7 830 8 23 37198 Unknown 24 8 1287 9 24 40171 Unknown 25 9 1975 10 25 42638 Unknown 26 10 2744 11 26 44653 Unknown 27 11 4515 12 27 46472 58761 28 12 5974 13 28 48467 63851 29 13 7711 14 29 49970 66492 30 14 9692 ----31 15 11791 ----\n\nThe SIR model for an infectious disease can be written as follows [6, 7] :\n\n,\n\nThe number of susceptible persons is S, infected (persons who are sick and spread the infection) -I, removed (persons who do not spread the infection anymore, this number is the sum of isolated, recovered and dead people) -R; the infection and immunization rates are  and  respectively.", "The sum of \"tested confirmed cases\" and \"clinically diagnosed cases\" Wj + Qj 16 0 45 1 16 14380 Unknown 17 1 62 2 17 17205 Unknown 18 2 121 3 18 20440 Unknown 19 3 198 4 19 24324 Unknown 20 4 291 5 20 28018 Unknown 21 5 440 6 21 31161 Unknown 22 6 571 7 22 34568 Unknown 23 7 830 8 23 37198 Unknown 24 8 1287 9 24 40171 Unknown 25 9 1975 10 25 42638 Unknown 26 10 2744 11 26 44653 Unknown 27 11 4515 12 27 46472 58761 28 12 5974 13 28 48467 63851 29 13 7711 14 29 49970 66492 30 14 9692 ----31 15 11791 ----\n\nThe SIR model for an infectious disease can be written as follows [6, 7] :\n\n,\n\nThe number of susceptible persons is S, infected (persons who are sick and spread the infection) -I, removed (persons who do not spread the infection anymore, this number is the sum of isolated, recovered and dead people) -R; the infection and immunization rates are  and  respectively. It follows from (1) and (2) that\n\nIntegration of (5) with the initial conditions (4) yields:\n\nFunction I has a maximum at S  and tends to zero at infinity, see [6, 7] . In comparison, the number of susceptible persons at infinity 0, S   and can be calculated with the use of (6) from a non-linear equation \n\nyields:\n\nThus, for every set of parameters N, ,  ,  0 t and a fixed value of V the integral (10) can be calculated and the corresponding moment of time can be determined from (9) .", "In comparison, the number of susceptible persons at infinity 0, S   and can be calculated with the use of (6) from a non-linear equation \n\nyields:\n\nThus, for every set of parameters N, ,  ,  0 t and a fixed value of V the integral (10) can be calculated and the corresponding moment of time can be determined from (9) . Then I can be calculated from (6) by putting S = N  V and function R from\n\nStatistical approach for parameter identification. Linear regression As in paper [4] , we shall use the fact that the random function 1 ( , , ) F V N  has a linear distribution (see (9) ).", "Linear regression As in paper [4] , we shall use the fact that the random function 1 ( , , ) F V N  has a linear distribution (see (9) ). Then we can apply the linear regression (see [10] ) for every pair of parameters N and  and calculate the corresponding values of 0 t and .  The optimal (the most reliable) values of N and  correspond to the maximum value of the correlation coefficient r (see [4, 9] ).", " The optimal (the most reliable) values of N and  correspond to the maximum value of the correlation coefficient r (see [4, 9] ). Since we did not know and still don't know the values of Qj before February 12, 2020, we supposed that Vj = Wj and have done the calculations with the use of data for the time period from January 16 to February 9, 2020. The optimal values of the parameters are:\n\nThe corresponding correlation coefficient is very high r = 0.997966487046645.", "The optimal values of the parameters are:\n\nThe corresponding correlation coefficient is very high r = 0.997966487046645. The solution of (7) yields the value 45579. The corresponding number of infected I, susceptible S and removed R persons versus time (starting from January 16, 2020) were calculated and shown in Fig. 2 .", "2 . The blue line represents the number of victims V = I + R and is in good agreement with \"tested confirmed cases\" Wj, reported by the National Health Commission of the People's Republic of China [1] (blue markers). Unfortunately, many cases have not been included in the official counts and have appeared in the official Table from [1] only on February 12 as \"clinically diagnosed cases\" Qj (see Fig.", "Unfortunately, many cases have not been included in the official counts and have appeared in the official Table from [1] only on February 12 as \"clinically diagnosed cases\" Qj (see Fig. 1 ). Since the National Health Commission of the the People's Republic of China has proposed two different ways of registration of the same disease [1] , Vj must be the sum of Wj and Qj , i.e.", "Since the National Health Commission of the the People's Republic of China has proposed two different ways of registration of the same disease [1] , Vj must be the sum of Wj and Qj , i.e. Vj = Wj + Qj (provided that no new methods of registering the same disease would appear). Values Wj after February 9 are shown in Fig.", "Values Wj after February 9 are shown in Fig. 3 by \"stars\". \"Crosses\" represent the sum Wj + Qj .", "3 by \"stars\". \"Crosses\" represent the sum Wj + Qj . Since the optimal curve was obtained only with the use of Wj and the difference between Wj and Vj is very big (e.g., it was 12 289 persons on February 12, 2020), the predictions shown in Fig.", "Since the optimal curve was obtained only with the use of Wj and the difference between Wj and Vj is very big (e.g., it was 12 289 persons on February 12, 2020), the predictions shown in Fig. 2 and reported in [11] are no longer relevant. To have better predictions, it is necessary to have exact Qjdata for the period before February 12.", "To have better predictions, it is necessary to have exact Qjdata for the period before February 12. Blue markers show the \"tested confirmed cases\" W j , reported by the National Health Commission of the People's Republic of China [1] . The \"circles\" correspond to the points used for calculations (it was supposed that V j = W j ); \"stars\" -to the points used only for verification Circles\" show the \"tested confirmed cases\" W j for the period from January 16 to February 9, 2020, [2] .", "The \"circles\" correspond to the points used for calculations (it was supposed that V j = W j ); \"stars\" -to the points used only for verification Circles\" show the \"tested confirmed cases\" W j for the period from January 16 to February 9, 2020, [2] . These points were used to calculate the prediction curve. \"Stars\" correspond to the \"tested confirmed cases\" W j for the period from February 10 to February 14, 2020, [1] .", "\"Stars\" correspond to the \"tested confirmed cases\" W j for the period from February 10 to February 14, 2020, [1] . \"Crosses\" represent the sum W j + Q j from [1] \n\nThe simple mathematical model was used to predict the characteristics of the epidemic caused by coronavirus in mainland China. The numbers of infected, susceptible, and removed persons versus time were predicted and compared with the new data obtained after February 10, 2020, when the calculations were completed.", "The numbers of infected, susceptible, and removed persons versus time were predicted and compared with the new data obtained after February 10, 2020, when the calculations were completed. Unfortunately, many cases have not been included in the official counts and have appeared on February 12 only. It makes the predictions reported on February 10, 2020, no longer relevant.", "It makes the predictions reported on February 10, 2020, no longer relevant. Further research should focus on updating the predictions with the use of corrected data and more complicated mathematical models." ]

[ "Defining the connections among neurons is critical to our understanding of the structure and function of the nervous system. Recombinant viruses engineered to transmit across synapses provide a powerful approach for the dissection of neuronal circuitry in vivo. We recently demonstrated that recombinant vesicular stomatitis virus (VSV) can be endowed with anterograde or retrograde transsynaptic tracing ability by providing the virus with different glycoproteins.", "We recently demonstrated that recombinant vesicular stomatitis virus (VSV) can be endowed with anterograde or retrograde transsynaptic tracing ability by providing the virus with different glycoproteins. Here we extend the characterization of the transmission and gene expression of recombinant VSV (rVSV) with the rabies virus glycoprotein (RABV-G), and provide examples of its activity relative to the anterograde transsynaptic tracer form of rVSV. rVSV with RABV-G was found to drive strong expression of transgenes and to spread rapidly from neuron to neuron in only a retrograde manner.", "rVSV with RABV-G was found to drive strong expression of transgenes and to spread rapidly from neuron to neuron in only a retrograde manner. Depending upon how the RABV-G was delivered, VSV served as a polysynaptic or monosynaptic tracer, or was able to define projections through axonal uptake and retrograde transport. In animals co-infected with rVSV in its anterograde form, rVSV with RABV-G could be used to begin to characterize the similarities and differences in connections to different areas.", "In animals co-infected with rVSV in its anterograde form, rVSV with RABV-G could be used to begin to characterize the similarities and differences in connections to different areas. rVSV with RABV-G provides a flexible, rapid, and versatile tracing tool that complements the previously described VSV-based anterograde transsynaptic tracer. Text: Mapping neuronal connectivity in the central nervous system (CNS) of even simple organisms is a difficult task.", "Text: Mapping neuronal connectivity in the central nervous system (CNS) of even simple organisms is a difficult task. Recombinant viruses engineered to trace synaptic connections and express transgenes promise to enable higher-throughput mapping of connections among neurons than other methods, e.g., serial reconstruction from electron micrographs (Bock et al., 2011; Briggman et al., 2011) . The Pseudorabies (PRV) and Rabies viruses (RABV) have been the best characterized and most utilized circuit tracing viruses to date (Ugolini et al., 1989; Kelly and Strick, 2000) .", "The Pseudorabies (PRV) and Rabies viruses (RABV) have been the best characterized and most utilized circuit tracing viruses to date (Ugolini et al., 1989; Kelly and Strick, 2000) . RABV was recently modified by Wickersham and colleagues such that it can travel across only one synapse, allowing for a straightforward definition of monosynaptic connections (Wickersham et al., 2007b) . This strategy permitted the first unambiguous identification of retrogradely connected cells from an initially infected cell (\"starter cell\"), without the need for electrophysiology.", "This strategy permitted the first unambiguous identification of retrogradely connected cells from an initially infected cell (\"starter cell\"), without the need for electrophysiology. Moreover, the starter cell could be defined through the expression of a specific viral receptor that limited the initial infection. Recently, we created an anterograde monosynaptic virus that complements the previously available retrograde viral tracers (Beier et al., 2011) .", "Recently, we created an anterograde monosynaptic virus that complements the previously available retrograde viral tracers (Beier et al., 2011) . Vesicular stomatitis virus (VSV), a virus related to RABV, with its own glycoprotein (G) gene (VSV-G), or with a G from the unrelated lymphocytic choriomeningitis virus (LCMV-G), spreads in the anterograde direction across synapses. VSV can be used as a polysynaptic tracer that spreads across many synapses, owing to the fact that the normal, replicationcompetent form of the virus does not cause serious diseases in humans (Brandly and Hanson, 1957; Johnson et al., 1966; Brody et al., 1967) .", "VSV can be used as a polysynaptic tracer that spreads across many synapses, owing to the fact that the normal, replicationcompetent form of the virus does not cause serious diseases in humans (Brandly and Hanson, 1957; Johnson et al., 1966; Brody et al., 1967) . Whether the virus is a monosynaptic or polysynaptic tracer is determined by the method of delivery of the G gene ( Figure 1A) . Advantages of VSV are that it is well-characterized, is relatively simple in comparison to PRV, and it rapidly grows to high titer in tissue culture cells.", "Advantages of VSV are that it is well-characterized, is relatively simple in comparison to PRV, and it rapidly grows to high titer in tissue culture cells. It is also being developed as a vaccine vector, often using a G of another virus as the immunogen, as well as being developed as a cytocidal agent that will target tumor cells in humans (Balachandran and Barber, 2000; Stojdl et al., 2000 Stojdl et al., , 2003 . Previous studies of the anatomical patterns of transmission, as well as physiological recordings, have shown that the transmission of VSV and RABV among neurons is via synapses (Kelly and Strick, 2000; Wickersham et al., 2007b; Beier et al., 2011) .", "Previous studies of the anatomical patterns of transmission, as well as physiological recordings, have shown that the transmission of VSV and RABV among neurons is via synapses (Kelly and Strick, 2000; Wickersham et al., 2007b; Beier et al., 2011) . In addition, it has been shown that RABV, as well as lentiviruses with RABV-G in their envelope, travel retrogradely from an injection site (Mazarakis et al., 2001; Wickersham et al., 2007a) . We hypothesized that providing a recombinant VSV (rVSV) with the RABV-G would create a retrograde polysynaptic transsynaptic tracer without the biosafety concerns inherent to RABV.", "We hypothesized that providing a recombinant VSV (rVSV) with the RABV-G would create a retrograde polysynaptic transsynaptic tracer without the biosafety concerns inherent to RABV. Our initial characterization of rVSV with RABV-G showed that indeed FIGURE 1 | Synaptic tracing strategies using VSV. (A) Schematic illustrating the strategies for polysynaptic or monosynaptic retrograde or anterograde transsynaptic transmission of rVSV encoding GFP.", "(A) Schematic illustrating the strategies for polysynaptic or monosynaptic retrograde or anterograde transsynaptic transmission of rVSV encoding GFP. The initially infected cell is indicated by an asterisk. VSV encoding a glycoprotein (G) within its genome can spread polysynaptically. The direction of the spread depends on the identity of the glycoprotein.", "The direction of the spread depends on the identity of the glycoprotein. Infected neurons are shown in green. In some cases, the initially infected starter cell can be defined by the expression of an avian receptor, TVA (tagged with a red fluorescent protein).", "In some cases, the initially infected starter cell can be defined by the expression of an avian receptor, TVA (tagged with a red fluorescent protein). The TVA-expressing neurons can then be specifically infected by rVSV G with the EnvA/RABV-G (A/RG) glycoprotein (Wickersham et al., 2007b) on the virion surface [rVSV G(A/RG)]. These starter cells are then yellow, due to viral GFP and mCherry from TVA-mCherry expression.", "These starter cells are then yellow, due to viral GFP and mCherry from TVA-mCherry expression. For monosynaptic tracing, the G protein is expressed in trans in the TVA-expressing cell, and thus complements rVSV G to allow transmission in a specific direction. (B) Genomic diagrams of rVSV vectors.", "(B) Genomic diagrams of rVSV vectors. All VSVs contain four essential proteins: N, P, M, and L. Some viruses encode a G gene in their genome, which allows them to spread polysynaptically. rVSV vectors typically encode a transgene in the first position, while others carry an additional transgene in the G position.", "rVSV vectors typically encode a transgene in the first position, while others carry an additional transgene in the G position. (C) Morphological characterization of rVSV-infected neurons in several locations within the mouse brain. (i,ii) Caudate-putamen (CP) neurons at 4 dpi from an injection of the CP with rVSV(VSV-G) viruses encoding (i) CFP or (ii) Korange.", "(i,ii) Caudate-putamen (CP) neurons at 4 dpi from an injection of the CP with rVSV(VSV-G) viruses encoding (i) CFP or (ii) Korange. (iii) Labeled neurons of the CA1 region of the hippocampus are shown at 5 dpi following injection into the hippocampus of rVSV(VSV-G) encoding Venus. (iv,v) Cortical pyramidal neurons are shown following injection into the CP of rVSV(RABV-G) expressing (iv) GFP at 24 hpi, or (v) mCherry at 48 hpi.", "(iv,v) Cortical pyramidal neurons are shown following injection into the CP of rVSV(RABV-G) expressing (iv) GFP at 24 hpi, or (v) mCherry at 48 hpi. Inset in (iv) is a high magnification of the neuron in panel (iv), highlighting labeling of dendritic spines. (vi) Multiple viruses can be co-injected into the same animal.", "(vi) Multiple viruses can be co-injected into the same animal. Here, individual rVSV G(VSV-G) viruses encoding CFP, GFP , Venus, Korange, and mCherry were used to infect the cortex. Scale bars = 50 µm.", "Scale bars = 50 µm. February 2013 | Volume 7 | Article 11 | 2 it could be taken up as a retrograde tracer (Beier et al., 2011) . To determine if it could transmit among neurons following its replication in neurons, and to further analyze the transmission patterns of both the monosynaptic and polysynaptic forms of rVSV with RABV-G, we made injections into several CNS and peripheral locations.", "To determine if it could transmit among neurons following its replication in neurons, and to further analyze the transmission patterns of both the monosynaptic and polysynaptic forms of rVSV with RABV-G, we made injections into several CNS and peripheral locations. In addition, we performed co-infections of rVSV with RABV-G and the anterograde form of rVSV in order to exploit the differences in the directionality of transmission of these two viruses in mapping circuits. Schematics of viruses created and used throughout this study are shown in Figure 1 .", "Schematics of viruses created and used throughout this study are shown in Figure 1 . We created rVSV vector plasmids carrying different transgenes in either the first or fifth genomic positions ( Figure 1B) . After rescuing each virus, we tested the ability of each to express transgenes in different brain regions through intracranial injections ( Figure 1C ).", "After rescuing each virus, we tested the ability of each to express transgenes in different brain regions through intracranial injections ( Figure 1C ). All rVSV vectors drove robust fluorophore expression 1 or 2 days post-infection (hpi) ( Figure 1C ) (van den Pol et al., 2009) . In fact, by 12 hpi, labeling was sufficiently bright to image fine morphological details, such as dendritic spines ( Figure 1C ,iv).", "In fact, by 12 hpi, labeling was sufficiently bright to image fine morphological details, such as dendritic spines ( Figure 1C ,iv). To characterize the physiological properties of cells infected with rVSV, we tested a replication-competent rVSV encoding GFP, with RABV-G in the genome in place of VSV-G [hereafter designated rVSV(RABV-G)]. van den Pol et al.", "van den Pol et al. reported that hippocampal neurons infected with replication-incompetent (G-deleted or \" G\") rVSV were physiologically healthy at 12-14 hpi, but were less so by 1 day post-infection (dpi) (van den Pol et al., 2009) . Given the known toxicity of both VSV and RABV-G (Coulon et al., 1982) , we tested the physiology of cortical pyramidal neurons in the motor cortex (M1) infected with rVSV(RABV-G).", "Given the known toxicity of both VSV and RABV-G (Coulon et al., 1982) , we tested the physiology of cortical pyramidal neurons in the motor cortex (M1) infected with rVSV(RABV-G). Between 12 and 18 hpi, the membrane capacitance, input resistance, resting membrane potential, and current-to-action potential firing relationship were indistinguishable between infected and uninfected neurons (Figure 2) . However, by 2 dpi, electrophysiological properties were so abnormal in the infected cortical pyramidal cells that physiological measurements could not be made.", "However, by 2 dpi, electrophysiological properties were so abnormal in the infected cortical pyramidal cells that physiological measurements could not be made. The speed and strength of the expression of transgenes encoded by VSV depends upon the gene's genomic position (van den Pol et al., 2009; Beier et al., 2011) . Genes in the first position are expressed the most highly, with a decrease in the level of expression in positions more 3 within the viral plus strand.", "Genes in the first position are expressed the most highly, with a decrease in the level of expression in positions more 3 within the viral plus strand. When GFP was inserted into the first position of VSV, GFP fluorescence was first detectable at approximately 1 hpi in cultured cells (van den Pol et al., 2009) . In order to quantify the relative expression of a fluorescent protein in the first genomic position in neurons, rat hippocampal slices were infected with a replication-incompetent rVSV that expresses mCherry (rVSV G, Figures 1A,B) .", "In order to quantify the relative expression of a fluorescent protein in the first genomic position in neurons, rat hippocampal slices were infected with a replication-incompetent rVSV that expresses mCherry (rVSV G, Figures 1A,B) . This was a G virus which had the RABV-G supplied in trans during the preparation of the virus stock [referred to as rVSV G(RABV-G)]. Average fluorescence intensity of the infected cells was measured every hour over the course of 18 h. By 4 hpi at 37 • C, red fluorescence was clearly visible, and reached maximal levels by approximately 14 hpi (N = 3, Figure 3 ).", "Average fluorescence intensity of the infected cells was measured every hour over the course of 18 h. By 4 hpi at 37 • C, red fluorescence was clearly visible, and reached maximal levels by approximately 14 hpi (N = 3, Figure 3 ). Similar results were obtained with a virus encoding GFP in the first genomic position rather than mCherry (i.e., Figure 1B ) (N = 3). We previously demonstrated that rVSV(RABV-G) could be taken up retrogradely by neurons (Beier et al., 2011) , but these experiments did not distinguish between direct axonal uptake of the initial inoculum vs. retrograde transsynaptic transmission following viral replication.", "We previously demonstrated that rVSV(RABV-G) could be taken up retrogradely by neurons (Beier et al., 2011) , but these experiments did not distinguish between direct axonal uptake of the initial inoculum vs. retrograde transsynaptic transmission following viral replication. To distinguish between these two mechanisms and to extend the previous analyses, we conducted further experiments in the mammalian visual system (Figures 4A-G) . As visual cortex area 1 (V1) does not receive direct projections from retinal ganglion cells (RGCs), but rather receives secondary input from RGCs via the lateral geniculate nucleus (LGN), infection of RGCs from injection of V1 would demonstrate retrograde transmission from cells which supported at least one round of viral replication.", "As visual cortex area 1 (V1) does not receive direct projections from retinal ganglion cells (RGCs), but rather receives secondary input from RGCs via the lateral geniculate nucleus (LGN), infection of RGCs from injection of V1 would demonstrate retrograde transmission from cells which supported at least one round of viral replication. Following a V1 injection with rVSV(RABV-G), GFP-positive RGCs were observed in the retina by 3 dpi (N = 3; Figure 4G ). Importantly, viral labeling in the brain was restricted to primary and secondary projection areas, even at 7 dpi.", "Importantly, viral labeling in the brain was restricted to primary and secondary projection areas, even at 7 dpi. These included the LGN ( Figure 4D ) and the hypothalamus (Figure 4E) , two areas known to project directly to V1 (Kandel, 2000) . Selective labeling was observed in other areas, such as cortical areas surrounding V1 (Figure 4C) , which project directly to V1, and also in the superior colliculus (SC) stratum griseum centrale, which projects to the LGN ( Figure 4F) .", "Selective labeling was observed in other areas, such as cortical areas surrounding V1 (Figure 4C) , which project directly to V1, and also in the superior colliculus (SC) stratum griseum centrale, which projects to the LGN ( Figure 4F) . Labeling was also observed in the nucleus basalis, which projects to the cortex, as well as many components of the basal ganglia circuit, which provide input to the thalamus [such as the caudate-putamen (CP), globus pallidus (GP), and the subthalamic nucleus (STn)]. The amygdala, which projects to the hypothalamus, was also labeled.", "The amygdala, which projects to the hypothalamus, was also labeled. Consistent with a lack of widespread viral transmission, animals did not exhibit signs of disease at 7 dpi. These data show that rVSV(RABV-G) can spread in a retrograde direction from the injection site, but do not address whether the virus can spread exclusively in the retrograde direction.", "These data show that rVSV(RABV-G) can spread in a retrograde direction from the injection site, but do not address whether the virus can spread exclusively in the retrograde direction. Directional transsynaptic specificity can only be definitively addressed using a unidirectional circuit. We therefore turned to the primary motor cortex (M1) to CP connection, in which neurons project from the cortex to the CP, but not in the other direction ( Figure 4H ) (Beier et al., 2011) .", "We therefore turned to the primary motor cortex (M1) to CP connection, in which neurons project from the cortex to the CP, but not in the other direction ( Figure 4H ) (Beier et al., 2011) . Injections of rVSV(RABV-G) into M1 should not label neurons in the CP if the virus can only label cells across synapses in the retrograde direction. Indeed, at 2 dpi, areas directly projecting to the injection site, including the contralateral cortex, were labeled ( Figure 4I ).", "Indeed, at 2 dpi, areas directly projecting to the injection site, including the contralateral cortex, were labeled ( Figure 4I ). Only axons from cortical cells were observed in the CP, with no GFP-labeled cell bodies present in the CP (Figure 4J) , consistent with lack of anterograde transsynaptic spread. By 3 dpi, a small number of medium spiny neurons (MSNs) in the CP were observed, likely via secondary spread from initially infected thalamic or GP neurons (data not shown).", "By 3 dpi, a small number of medium spiny neurons (MSNs) in the CP were observed, likely via secondary spread from initially infected thalamic or GP neurons (data not shown). A particular advantage of retrograde viral tracers is the ability to label CNS neurons projecting to peripheral sites. This has been a powerful application of both RABV and PRV (Ugolini et al., 1989; Standish et al., 1994) .", "This has been a powerful application of both RABV and PRV (Ugolini et al., 1989; Standish et al., 1994) . To test if rVSV(RABV-G) could also perform this function, we examined the innervation of the dura surface by neurons of the trigeminal ganglion, a neuronal circuit thought to be involved in migraine headaches (Penfield and McNaughton, 1940; Mayberg et al., 1984) . These neurons have axons, but not canonical dendrites, and send projections into the spinal cord and brainstem.", "These neurons have axons, but not canonical dendrites, and send projections into the spinal cord and brainstem. Therefore, the only way trigeminal neurons could become labeled from viral application to the dura is through retrograde uptake of the virus. We applied rVSV(RABV-G) to the intact dura mater and analyzed the dura, trigeminal ganglion, and CNS for labeling ( Figure 4K ).", "We applied rVSV(RABV-G) to the intact dura mater and analyzed the dura, trigeminal ganglion, and CNS for labeling ( Figure 4K ). At the earliest time point examined, 3 dpi, we observed axons traveling along the dura, but little other evidence of infection ( Figure 4L) . No labeled neuronal cell bodies on the dura were observed, consistent with the lack of neurons on this surface.", "No labeled neuronal cell bodies on the dura were observed, consistent with the lack of neurons on this surface. In contrast, we did find labeled cell bodies in the trigeminal ganglion ( Figure 4M ). No infection was seen in the CNS, even at 4 dpi, consistent with the lack of inputs from the brain into the trigeminal ganglion (N = 4 animals).", "No infection was seen in the CNS, even at 4 dpi, consistent with the lack of inputs from the brain into the trigeminal ganglion (N = 4 animals). To further characterize patterns and kinetics of viral transmission and directional specificity of transsynaptic spread, injections of rVSV(RABV-G) were made into the CP ( Figure 5A ). In order to determine which cells were labeled by direct uptake of virus in the inoculum, a separate set of animals were injected into the CP with the replication-incompetent rVSV G(RABV-G) (N = 3 animals, analyzed 3 dpi).", "In order to determine which cells were labeled by direct uptake of virus in the inoculum, a separate set of animals were injected into the CP with the replication-incompetent rVSV G(RABV-G) (N = 3 animals, analyzed 3 dpi). Cells labeled by rVSV G(RABV-G) were observed in the CP, GP, substantia nigra (SN), thalamus, and layers 3 and 5 of the cortex, consistent with infection at the axon terminal and retrograde labeling of cell bodies of neurons known to project directly to the CP ( Figure 5C ) (Albin et al., 1995) . Areas labeled by CP injection are indicated in Figure 5B .", "Areas labeled by CP injection are indicated in Figure 5B . The patterns of spread for the replication-competent rVSV(RABV-G) were characterized over the course of 1-5 dpi ( Figures 5D-H) . During this interval, progressively more cells in infected regions were labeled by rVSV(RABV-G), including within the CP, nucleus basalis, cortex, and GP (listed in Figure 5B ).", "During this interval, progressively more cells in infected regions were labeled by rVSV(RABV-G), including within the CP, nucleus basalis, cortex, and GP (listed in Figure 5B ). In addition, more cortical cells were labeled in clusters near cortical pyramidal neurons, both ipsilateral and contralateral to the injected side, including neurogliaform cells (data not shown). These data are in contrast to those observed following infection with an anterograde transsynaptic tracing virus, such as rVSV with its own G gene, rVSV(VSV-G) (Figure 5B) .", "These data are in contrast to those observed following infection with an anterograde transsynaptic tracing virus, such as rVSV with its own G gene, rVSV(VSV-G) (Figure 5B) . At 3 dpi following rVSV(VSV-G) injection into the CP, the cerebral cortex was not labeled, but regions receiving projections from the CP, such as the STn, GP, and SN, were labeled (Beier et al., 2011) . In order to investigate other areas for evidence of cell-to-cell retrograde transsynaptic spread, the nucleus basalis was examined following infection of the CP with replication-competent rVSV(RABV-G).", "In order to investigate other areas for evidence of cell-to-cell retrograde transsynaptic spread, the nucleus basalis was examined following infection of the CP with replication-competent rVSV(RABV-G). The nucleus basalis was labeled by 2 dpi (Figures 5E-H) , consistent with at least a single transsynaptic jump, as this area does not directly project to the CP. The virus appeared to travel transsynaptically at the rate of roughly 1 synapse per day, as evidenced by the lack of labeled neurogliaform cells in the cortex, and lack of neurons in the nucleus basalis at 1 dpi, and label appearing in these cell types/areas at 2 dpi, as previously observed (Beier et al., 2011) .", "The virus appeared to travel transsynaptically at the rate of roughly 1 synapse per day, as evidenced by the lack of labeled neurogliaform cells in the cortex, and lack of neurons in the nucleus basalis at 1 dpi, and label appearing in these cell types/areas at 2 dpi, as previously observed (Beier et al., 2011) . Labeling remained well-restricted to the expected corticostriatal circuits at 5 dpi, suggesting that viral spread becomes less efficient after crossing one or two connections, consistent with injections into V1 (Figure 4) . While glial cells can be infected and were observed near the injection site (van den Pol et al., 2002; Chauhan et al., 2010) , infected glial cells away from the injection site generally were not observed.", "While glial cells can be infected and were observed near the injection site (van den Pol et al., 2002; Chauhan et al., 2010) , infected glial cells away from the injection site generally were not observed. One advantage of having both anterograde and retrograde forms of the same virus is that they can be used in parallel, or in tandem, to trace circuitry to and from a single or multiple sites of injection, with each virus having similar kinetics of spread and gene expression. In fact, if different fluorophores are used in different viruses, e.g., rVSV(VSV-G) and rVSV(RABV-G), then the viruses can be co-injected into the same site and their transmission can be traced independently ( Figure 6A ).", "In fact, if different fluorophores are used in different viruses, e.g., rVSV(VSV-G) and rVSV(RABV-G), then the viruses can be co-injected into the same site and their transmission can be traced independently ( Figure 6A ). This is most straightforward if there are no cells at the injection site that are initially infected by both viruses. Co-infected cells can be easily detected, as they would express both fluorescent proteins shortly after injection.", "Co-infected cells can be easily detected, as they would express both fluorescent proteins shortly after injection. In order to determine whether two viruses would allow simultaneous anterograde and retrograde transsynaptic tracing from a single injection site, a rVSV(VSV-G) expressing Venus and a rVSV(RABV-G) expressing mCherry were injected individually (Figures 6B-D) or co-injected (Figures 6E-G) into the motor cortex, and brains were examined 3 dpi. The pattern of labeling from the co-injected brains was equivalent to the patterns observed when each virus was injected individually: rVSV(VSV-G) was observed to infect neurons in the cortex, CP, and downstream nuclei, whereas the rVSV(RABV-G) was not observed to infect neurons in the CP, but rather in the thalamus and nucleus basalis (N = 4).", "The pattern of labeling from the co-injected brains was equivalent to the patterns observed when each virus was injected individually: rVSV(VSV-G) was observed to infect neurons in the cortex, CP, and downstream nuclei, whereas the rVSV(RABV-G) was not observed to infect neurons in the CP, but rather in the thalamus and nucleus basalis (N = 4). The initial co-infection rate is dependent upon The presence or absence of labeling is indicated by (+) and (−), respectively. The extent of labeling is indicated by the number of (+).", "The extent of labeling is indicated by the number of (+). Some animals were infected with G viruses to determine which areas were labeled by direct uptake of the virions, rather than by replication and transmission. These were sacrificed at 3 dpi.", "These were sacrificed at 3 dpi. (C) Parasaggital section of a brain infected with VSV[greek delta]G(RABV-G). The injection site is marked by a red arrow.", "The injection site is marked by a red arrow. Several areas that project directly to the CP were labeled due to direct uptake of the virions, including the cortex, thalamus, and GP (arrowheads), 3 dpi. the dose of the initial inocula.", "the dose of the initial inocula. When injecting 3 × 10 3 focus forming units (ffu) rVSV(VSV-G) and 3 × 10 4 ffu rVSV(RABV-G), no co-infection was observed at the injection site. Thus, co-infection of the same brain region, without co-infection of the same cells,\n\ndoes not alter the spreading behavior of either rVSV(VSV-G) or rVSV(RABV-G).", "Thus, co-infection of the same brain region, without co-infection of the same cells,\n\ndoes not alter the spreading behavior of either rVSV(VSV-G) or rVSV(RABV-G). One example of how this dual retrograde and anterograde transsynaptic tracing system can be used is to determine if three Frontiers in Neural Circuits February 2013 | Volume 7 | Article 11 | 8 distinct regions are connected and the directionality of any connections. For example, the anterograde transsynaptic virus can be injected into one region, the retrograde into another, and a third region can then be examined for evidence of labeling by either or both viruses (e.g., Figure 6H ).", "For example, the anterograde transsynaptic virus can be injected into one region, the retrograde into another, and a third region can then be examined for evidence of labeling by either or both viruses (e.g., Figure 6H ). To test this possibility, rVSV(VSV-G) was injected into the motor cortex, rVSV(RABV-G) was injected into the substantia nigra pars reticulara (SNr), and animals were sacrificed at 3 dpi. We observed that cells were singly labeled, either with Venus [rVSV(VSV-G)] or with mCherry [rVSV(RABV-G)], and were located largely in different regions of the CP (Figures 6I,J) (N = 3) .", "We observed that cells were singly labeled, either with Venus [rVSV(VSV-G)] or with mCherry [rVSV(RABV-G)], and were located largely in different regions of the CP (Figures 6I,J) (N = 3) . These results suggest that the anterograde connections from the cells infected with rVSV(VSV-G) in the M1 were with CP MSNs that did not project to the region of the SNr injected with rVSV(RABV-G) (N = 3 animals). In addition to polysynaptic tracing, VSV can be modified to trace circuits monosynaptically (Beier et al., 2011) .", "In addition to polysynaptic tracing, VSV can be modified to trace circuits monosynaptically (Beier et al., 2011) . With RABV, this was achieved in vivo by first infecting with an adeno-associated virus (AAV) expressing TVA, a receptor for an avian retrovirus, and RABV-G (Wall et al., 2010) . This was followed 3 weeks later by infection with a G RABV with an EnvA/RABV-G chimeric glycoprotein on the virion surface (Wickersham et al., 2007b) , which allowed infection specifically of the cells expressing TVA.", "This was followed 3 weeks later by infection with a G RABV with an EnvA/RABV-G chimeric glycoprotein on the virion surface (Wickersham et al., 2007b) , which allowed infection specifically of the cells expressing TVA. A similar strategy was used to test rVSV's ability to monosynaptically trace retrogradely connected neurons in vivo. Inputs to choline acetyltransferase (ChAT)-expressing neurons in the striatum were used for this test.", "Inputs to choline acetyltransferase (ChAT)-expressing neurons in the striatum were used for this test. These neurons primarily receive input from the cortex and the thalamus (Thomas et al., 2000; Bloomfield et al., 2007) (Figure 7A) . In order to mark this population, we crossed ChAT-Cre mice to Ai9 mice, which express tdTomato in cells with a Cre expression history (Madisen et al., 2010) .", "In order to mark this population, we crossed ChAT-Cre mice to Ai9 mice, which express tdTomato in cells with a Cre expression history (Madisen et al., 2010) . Six-week-old mice from this cross were injected in the CP with two AAV vectors: one expressing a Cre-conditional (\"floxed\") TVA-mCherry fusion protein, and another expressing a floxed RABV-G. Two weeks later, the mice were injected in the same coordinates with rVSV G with the EnvA/RABV-G chimeric glycoprotein on the virion surface [rVSV G(A/RG)] (Beier et al., 2011) . Cells successfully infected with these two AAV vectors could host infection by a rVSV and should be able to produce rVSV virions with RABV-G on the surface.", "Cells successfully infected with these two AAV vectors could host infection by a rVSV and should be able to produce rVSV virions with RABV-G on the surface. Such starter cells should also express tdTomato and GFP. If rVSV were to be produced, and if it were to transmit across the synapse retrogradely, cortical and thalamic neurons should be labeled by GFP.", "If rVSV were to be produced, and if it were to transmit across the synapse retrogradely, cortical and thalamic neurons should be labeled by GFP. Mice injected with these AAV and rVSV viruses were sacrificed 5 days after rVSV infection, and brains analyzed for fluorescence. As expected for starter cells, some neurons in the CP expressed both tdTomato and GFP (Figure 7B) .", "As expected for starter cells, some neurons in the CP expressed both tdTomato and GFP (Figure 7B) . Outside of the CP, small numbers of GFP+ neurons that were not mCherry+ were observed in the cortex (Figures 7C,D) and thalamus (Figure 7E) , consistent with retrograde spread. Control animals not expressing Cre, or not injected with AAV encoding RABV-G, did not label cells in the cortex or thalamus (N = 3 for both controls and experimental condition).", "Control animals not expressing Cre, or not injected with AAV encoding RABV-G, did not label cells in the cortex or thalamus (N = 3 for both controls and experimental condition). Here, we report on the use of rVSV as a retrograde transsynaptic tracer for CNS circuitry. VSV can be modified to encode the RABV-G protein in the viral genome, allowing the virus to replicate and transmit across multiple synaptically connected cells, i.e., as a polysynaptic tracer.", "VSV can be modified to encode the RABV-G protein in the viral genome, allowing the virus to replicate and transmit across multiple synaptically connected cells, i.e., as a polysynaptic tracer. Alternatively, if the virus has the G gene deleted from its genome and RABV-G is provided in trans, it behaves as a monosynaptic tracer (Beier et al., 2011) . Although it has been known for many years that RABV travels retrogradely among neurons (Astic et al., 1993; Ugolini, 1995; Kelly and Strick, 2003) , and pseudotyping lentiviruses with RABV-G is sufficient for axonal transport (Mazarakis et al., 2001) , the retrograde transmission specificity among neurons had not been clearly shown to be a property of the G protein itself, as it might have been due to other viral proteins in addition to, or instead of, the viral G protein.", "Although it has been known for many years that RABV travels retrogradely among neurons (Astic et al., 1993; Ugolini, 1995; Kelly and Strick, 2003) , and pseudotyping lentiviruses with RABV-G is sufficient for axonal transport (Mazarakis et al., 2001) , the retrograde transmission specificity among neurons had not been clearly shown to be a property of the G protein itself, as it might have been due to other viral proteins in addition to, or instead of, the viral G protein. Since native VSV does not have these retrograde transsynaptic properties (van den Pol et al., 2002; Beier et al., 2011) , and the only alteration to the VSV genome was the substitution of the VSV G gene with the G gene of RABV, it is clear that the RABV glycoprotein is responsible for retrograde direction of viral transmission across synapses, at least in the case of rVSV. The early onset of gene expression from VSV relative to RABV (one hour vs. multiple hours) makes it beneficial in experimental paradigms in which the experiment needs to be done within a narrow window of time, such as tissue slices and explants.", "The early onset of gene expression from VSV relative to RABV (one hour vs. multiple hours) makes it beneficial in experimental paradigms in which the experiment needs to be done within a narrow window of time, such as tissue slices and explants. In addition, more than one transgene can be encoded in the viral genome without the need of a 2A or IRES element. The use of the first position of the genome enhances the expression level of the transgene inserted at that location, since VSV (and RABV) express genes in a transcriptional gradient; therefore, the first gene is the most highly transcribed (Knipe, 2007) .", "The use of the first position of the genome enhances the expression level of the transgene inserted at that location, since VSV (and RABV) express genes in a transcriptional gradient; therefore, the first gene is the most highly transcribed (Knipe, 2007) . This leads to rational predictions of expression levels so that one can choose the position of insertion of a transgene, or transgenes, according to this gradient and the desired level of expression. The size of the viral capsid is apparently not rigid, allowing for the inclusion of genomes that are substantially larger than the native genome, unlike the rigid capacity for some other viral vectors, such as AAV Yan et al., 2000) .", "The size of the viral capsid is apparently not rigid, allowing for the inclusion of genomes that are substantially larger than the native genome, unlike the rigid capacity for some other viral vectors, such as AAV Yan et al., 2000) . The fact that VSV can be made to spread anterogradely (Beier et al., 2011) or retrogradely across synapses with the change of a single gene affords several advantages over viral tracers that heretofore have not shown such flexibility in the directionality of tracing. In addition to the obvious application of tracing anterograde connections, combinations can be made to exploit the different forms of the virus.", "In addition to the obvious application of tracing anterograde connections, combinations can be made to exploit the different forms of the virus. One example that employs the simultaneous infection with an anterograde and retrograde form of VSV is demonstrated in Figure 6 . This experiment was designed to address whether the anterograde projections from the cortex to the CP would label the same brain regions as were labeled by a retrograde virus injected into the SN.", "This experiment was designed to address whether the anterograde projections from the cortex to the CP would label the same brain regions as were labeled by a retrograde virus injected into the SN. Although a block of superinfection by the virus may preclude infection of the same cell with multiple rVSVs, adjacent cells could still become labeled by different viruses (Whitaker-Dowling et al., 1983) . The observed results could be due to a preferential labeling by the anterograde transsynaptic virus of indirect pathway MSNs in this experiment, which then synapse onto the GP, thereby reflecting a viral bias.", "The observed results could be due to a preferential labeling by the anterograde transsynaptic virus of indirect pathway MSNs in this experiment, which then synapse onto the GP, thereby reflecting a viral bias. Alternatively, it could indicate that the cortical neurons in the injected region largely do not label the MSNs that project to the area of the SN injected with the retrograde virus. One further possibility is that too little virus was used to observe co-labeling of a given region.", "One further possibility is that too little virus was used to observe co-labeling of a given region. However, given the density of infection (i.e., Figures 6I,J) , the latter possibility seems unlikely. Additionally, the spread of the polysynaptic rVSV(RABV-G) appears to attenuate with increasing numbers of synapses crossed, permitting an analysis of more restricted viral spread.", "Additionally, the spread of the polysynaptic rVSV(RABV-G) appears to attenuate with increasing numbers of synapses crossed, permitting an analysis of more restricted viral spread. This is quite fortuitous, as if spread were to continue, it would lead to widespread infection and lethality. In addition, reconstruction of connectivity would be more difficult.", "In addition, reconstruction of connectivity would be more difficult. This reduced efficiency appears to also hold for the monosynaptic form of VSV complemented with RABV-G, as the efficiency of transmission appeared lower than the comparable experiment with RABV (Watabe-Uchida et al., 2012) . This is likely due to viral attenuation when VSV-G is replaced with RABV-G.\n\nWe were attracted to the use of VSV as a viral tracer due to its long track record as a safe, replication-competent laboratory agent.", "This is likely due to viral attenuation when VSV-G is replaced with RABV-G.\n\nWe were attracted to the use of VSV as a viral tracer due to its long track record as a safe, replication-competent laboratory agent. Laboratory workers using VSV have not contracted any diseases, and natural VSV infections among human populations in Central America and the southwestern United States (Rodríguez, 2002) occur without evident pathology (Johnson et al., 1966; Brody et al., 1967) . VSV was thus an attractive candidate for its use as a polysynaptic tracer for CNS studies, which requires an ability to replicate through multiple transmission cycles.", "VSV was thus an attractive candidate for its use as a polysynaptic tracer for CNS studies, which requires an ability to replicate through multiple transmission cycles. Both replicationcompetent and incompetent forms of VSV are in use under Biosafety Level 2 containment. Replication-competent RABV is Biosafety Level 3, due to the fact that infection with replicationcompetent RABV is almost always fatal to humans and in mice when infected intracerebrally (Smith, 1981; Knipe, 2007) .", "Replication-competent RABV is Biosafety Level 3, due to the fact that infection with replicationcompetent RABV is almost always fatal to humans and in mice when infected intracerebrally (Smith, 1981; Knipe, 2007) . Differences in pathogenicity between VSV and RABV are likely due to the ability of RABV to evade the innate immune system, particularly interferon (Hangartner et al., 2006; Junt et al., 2007; Lyles and Rupprecht, 2007; Rieder and Conzelmann, 2009; Iannacone et al., 2010) . VSV infection efficiently triggers an interferon response, and it has not evolved a method of escape from this response, unlike RABV (Brzózka et al., 2006) .", "VSV infection efficiently triggers an interferon response, and it has not evolved a method of escape from this response, unlike RABV (Brzózka et al., 2006) . In fact, VSV is being pursued as a vaccine for other viruses, including RABV (Lichty et al., 2004; Publicover et al., 2004; Kapadia et al., 2005; Schwartz et al., 2007; Iyer et al., 2009; Geisbert and Feldmann, 2011) . VSV does not typically spread beyond the initially infected site in the periphery (Kramer et al., 1983; Vogel and Fertsch, 1987) .", "VSV does not typically spread beyond the initially infected site in the periphery (Kramer et al., 1983; Vogel and Fertsch, 1987) . This likely is the cause of the minor or absent symptoms in humans and animals infected in nature. Polysynaptic VSV vectors are thus predicted to be much safer than polysynaptic RABV vectors.", "Polysynaptic VSV vectors are thus predicted to be much safer than polysynaptic RABV vectors. We have tested this prediction by injecting a series of mice in the footpads and hind leg muscles with rVSV(RABV-G), with the result that no injected animals showed any evidence of morbidity or mortality (Beier, Goz et al., in preparation) . While safer for laboratory workers than RABV, the main drawback to using VSV is its rapid cellular toxicity (van den Pol et al., 2009; Beier et al., 2011) .", "While safer for laboratory workers than RABV, the main drawback to using VSV is its rapid cellular toxicity (van den Pol et al., 2009; Beier et al., 2011) . Toxicity is due to suppression of cellular transcription and a block in the export of cellular RNAs from the nucleus to the cytoplasm (Black and Lyles, 1992; Her et al., 1997; Ahmed and Lyles, 1998; Petersen et al., 2000; von Kobbe et al., 2000) , as well as inhibition of the translation of cellular mRNAs (Francoeur et al., 1987; Jayakar et al., 2000; Kopecky et al., Frontiers in Neural Circuits February 2013 | Volume 7 | Article 11 | 10 2001). VSV is much quicker to enact its gene expression program than is RABV, such that cells suffer the toxic effects more quickly than after RABV infection.", "VSV is much quicker to enact its gene expression program than is RABV, such that cells suffer the toxic effects more quickly than after RABV infection. One aspect of VSV that can be exploited in the future to ameliorate the speed of toxicity is the use of VSV mutants and variants. One such mutant is the M51R, which permitted us to conduct physiological analyses of pre-and post-synaptic cells (Beier et al., 2011) .", "One such mutant is the M51R, which permitted us to conduct physiological analyses of pre-and post-synaptic cells (Beier et al., 2011) . We are in the process of examining the transmission properties of this mutant in vivo, as well as the effects of other mutations or viral variants on prolonging the health of neurons after infection. rVSV vectors can be used to study the connectivity of neuronal circuitry.", "rVSV vectors can be used to study the connectivity of neuronal circuitry. In addition to combinations of replication-competent forms of VSV, the replication-incompetent, monosynaptic forms of the virus can be easily combined, without the need to change viruses (Beier et al., 2011) . This allows a straightforward way to study both the projections into, and out from, a genetically defined cell population.", "This allows a straightforward way to study both the projections into, and out from, a genetically defined cell population. This can be done with the same viral genome, with the only change needed being the glycoprotein, for the selection of the direction of transmission. This flexibility of VSV makes it a powerful, multi-application vector for studying connectivity in the CNS.", "This flexibility of VSV makes it a powerful, multi-application vector for studying connectivity in the CNS. All rVSV clones were cloned from the rVSV G backbone (Chandran et al., 2005) . mCherry, Kusabira orange, Venus, and CFP were cloned into the first (GFP) position using XhoI and MscI sites, and VSV-G (a gift from Richard Mulligan, Harvard Medical School, Boston, MA) and RABV-G (a gift from Ed Callaway, Salk Institute, San Diego, CA) were cloned into the fifth (G) position using the MluI and NotI restriction sites.", "mCherry, Kusabira orange, Venus, and CFP were cloned into the first (GFP) position using XhoI and MscI sites, and VSV-G (a gift from Richard Mulligan, Harvard Medical School, Boston, MA) and RABV-G (a gift from Ed Callaway, Salk Institute, San Diego, CA) were cloned into the fifth (G) position using the MluI and NotI restriction sites. Genes for fluorescent proteins were obtained from Clontech. Viruses were rescued as previously described (Whelan et al., 1995) .", "Viruses were rescued as previously described (Whelan et al., 1995) . At 95% confluency, eight 10 cm plates of BSR cells were infected at an MOI of 0.01. Viral supernatants were collected at 24-h time intervals and ultracentrifuged at 21,000 RPM using a SW28 rotor and resuspended in 0.2% of the original volume.", "Viral supernatants were collected at 24-h time intervals and ultracentrifuged at 21,000 RPM using a SW28 rotor and resuspended in 0.2% of the original volume. For titering, concentrated viral stocks were applied in a dilution series to 100% confluent BSR cells and plates were examined at 12 hpi. Viral stocks were stored at −80 • C.\n\nFor G viruses, 293T cells were transfected with PEI (Ehrhardt et al., 2006) at 70% confluency on 10 cm dishes with 5 µg of pCAG-RABV-G. Twenty-four hours post-infection, the cells were infected at an MOI of 0.01 with rVSV G expressing either GFP or mCherry.", "Viral stocks were stored at −80 • C.\n\nFor G viruses, 293T cells were transfected with PEI (Ehrhardt et al., 2006) at 70% confluency on 10 cm dishes with 5 µg of pCAG-RABV-G. Twenty-four hours post-infection, the cells were infected at an MOI of 0.01 with rVSV G expressing either GFP or mCherry. Viral supernatants were collected for the subsequent 4 days at 24 h intervals. Virus preparations are now available from the Salk GT3 viral core ( All plasmids are available from Addgene (\n\nAAV-FLEx-RABV-G and AAV-FLEx-TVA-mCherry plasmids originated from the Lab of Naoshige Uchida (Watabe-Uchida et al., 2012) , and virus stocks were generous gifts from Brad Lowell, Harvard Medical School.", "Virus preparations are now available from the Salk GT3 viral core ( All plasmids are available from Addgene (\n\nAAV-FLEx-RABV-G and AAV-FLEx-TVA-mCherry plasmids originated from the Lab of Naoshige Uchida (Watabe-Uchida et al., 2012) , and virus stocks were generous gifts from Brad Lowell, Harvard Medical School. ChAT-Cre (B6;129S6-Chat tm1(cre)Lowl /J) and Ai9 (B6.Cg-Gt(ROSA)26Sor<tm9(CAG-tdTomato)Hze>/J) mice were obtained from the Jackson Laboratory (Madisen et al., 2010) . Eight-week-old CD-1 mice were injected using pulled capillary microdispensers (Drummond Scientific, Cat.", "Eight-week-old CD-1 mice were injected using pulled capillary microdispensers (Drummond Scientific, Cat. No: 5-000-2005) , using coordinates from The Mouse Brain in Stereotaxic Coordinates (Franklin and Paxinos, 1997) . Injection coordinates (in mm) used were: For multi-color analysis (Figures 1C,D) , 3 × 10 9 ffu/mL rVSV was injected into various regions.", "Injection coordinates (in mm) used were: For multi-color analysis (Figures 1C,D) , 3 × 10 9 ffu/mL rVSV was injected into various regions. For CP injections, 100 nL of rVSV(RABV-G) or rVSV(VSV-G) at 3 × 10 7 ffu/mL was injected at a rate of 100 nL/min. For the replication-incompetent viruses, 100 nL of 1 × 10 7 ffu/mL rVSV G(RABV-G) or rVSV G (VSV-G) was injected.", "For the replication-incompetent viruses, 100 nL of 1 × 10 7 ffu/mL rVSV G(RABV-G) or rVSV G (VSV-G) was injected. In the motor cortex, 100 nL of 1 × 10 7 ffu/mL rVSV(RABV-G) was injected, and mice harvested 2 dpi. For V1 injections, 100 nL of 3 × 10 10 ffu/mL rVSV(RABV-G) was injected, and mice were examined 3 or 7 dpi.", "For V1 injections, 100 nL of 3 × 10 10 ffu/mL rVSV(RABV-G) was injected, and mice were examined 3 or 7 dpi. For infections of the dura mater, 1 µL of 3 × 10 10 ffu/mL rVSV(RABV-G) was applied to the surface of the dura. The virus was allowed to absorb, and the surface was subsequently covered in bone wax, and the wound sutured.", "The virus was allowed to absorb, and the surface was subsequently covered in bone wax, and the wound sutured. For co-injections of virus into the same animal, 100 nL of a combination of 3 × 10 7 ffu/mL rVSV(VSV-G) and 3 × 10 8 ffu/mL rVSV(RABV-G) were co-injected into the motor cortex, and brains examined 3 dpi. For injections of the viruses into different regions, 100 nL of 3 × 10 7 ffu/mL rVSV(VSV-G) was injected into M1, and 100 nL of 3 × 10 8 ffu/mL rVSV(RABV-G) into the SNr, and brains examined 3 dpi.", "For injections of the viruses into different regions, 100 nL of 3 × 10 7 ffu/mL rVSV(VSV-G) was injected into M1, and 100 nL of 3 × 10 8 ffu/mL rVSV(RABV-G) into the SNr, and brains examined 3 dpi. A lower titer of rVSV(VSV-G) was used, as rVSV(RABV-G) is attenuated. All mouse work was conducted in biosafety containment level 2 conditions and was approved by the Longwood Medical Area Institutional Animal Care and Use Committee.", "All mouse work was conducted in biosafety containment level 2 conditions and was approved by the Longwood Medical Area Institutional Animal Care and Use Committee. Recordings were made from cortical pyramidal neurons in slices taken from postnatal day 12-18 mice, inoculated in the CP 12-18 h prior with rVSV(RABV-G). Coronal slices (300 µm thick) were cut in ice-cold external solution containing (in mM): 110 choline, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 2.5 KCl, 7 MgCl 2 , 0.5 CaCl 2 , 25 glucose, 11.6 Na-ascorbate, and 3.1 Na-pyruvate, bubbled with 95% O 2 and 5% CO 2 .", "Coronal slices (300 µm thick) were cut in ice-cold external solution containing (in mM): 110 choline, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 2.5 KCl, 7 MgCl 2 , 0.5 CaCl 2 , 25 glucose, 11.6 Na-ascorbate, and 3.1 Na-pyruvate, bubbled with 95% O 2 and 5% CO 2 . Slices were then transferred to artificial cerebrospinal fluid (ACSF) containing (in mM): 127 NaCl, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 2.5 KCl, 1 MgCl 2 , 2 CaCl 2 , and 25 glucose, bubbled with 95% O 2 and 5% CO 2 . After an incubation period Frontiers in Neural Circuits February 2013 | Volume 7 | Article 11 | 11 of 30-40 min at 34 • C, slices were stored at room temperature.", "After an incubation period Frontiers in Neural Circuits February 2013 | Volume 7 | Article 11 | 11 of 30-40 min at 34 • C, slices were stored at room temperature. All experiments were conducted at room temperature (25 • C). In all experiments, 50 µM picrotoxin, 10 µM 2,3-Dioxo-6-nitro-1, 2, 3, 4 -tetrahydrobenzo [f]quinoxaline -7-sulfonamide (NBQX), and 10 µM 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) were present in the ACSF to block GABAA/C, AMPA, and NMDA receptor-mediated transmission, respectively.", "In all experiments, 50 µM picrotoxin, 10 µM 2,3-Dioxo-6-nitro-1, 2, 3, 4 -tetrahydrobenzo [f]quinoxaline -7-sulfonamide (NBQX), and 10 µM 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) were present in the ACSF to block GABAA/C, AMPA, and NMDA receptor-mediated transmission, respectively. All chemicals were from Sigma or Tocris. Whole-cell recordings were obtained from infected and uninfected deep layer cortical pyramidal neurons identified with video-IR/DIC and GFP fluorescence was detected using epifluorescence illumination.", "Whole-cell recordings were obtained from infected and uninfected deep layer cortical pyramidal neurons identified with video-IR/DIC and GFP fluorescence was detected using epifluorescence illumination. With the deep layers of the cortex, 2-photon laser scanning microscopy (2PLSM) was used to confirm the cell types based on morphology. Deep layer pyramidal neurons had large cell bodies, classic pyramidal shape and dendritic spines.", "Deep layer pyramidal neurons had large cell bodies, classic pyramidal shape and dendritic spines. Glass electrodes (2-4 M ) were filled with internal solution containing (in mM): 135 KMeSO 4 , 5 KCl, 5 HEPES, 4 MgATP, 0.3 NaGTP, 10 Na 2 HPO 4 , 1 EGTA, and 0.01 Alexa Fluor-594 (to image neuronal morphology) adjusted to pH 7.4 with KOH. Current and voltage recordings were made at room temperature using a AxoPatch 200B or a Multiclamp 700B amplifier.", "Current and voltage recordings were made at room temperature using a AxoPatch 200B or a Multiclamp 700B amplifier. Data was filtered at 5 kHz and digitized at 10 kHz. Imaging and physiology data were acquired and analyzed as described previously (Carter and Sabatini, 2004) .", "Imaging and physiology data were acquired and analyzed as described previously (Carter and Sabatini, 2004) . Resting membrane potential was determined by the average of three 5-s sweeps with no injected current. Passive properties of the cell, membrane (Rm) and series resistance (Rs) and capacitance (Cm), were measured while clamping cells at −65 mV and applying voltage steps from −55 to −75 mV.", "Passive properties of the cell, membrane (Rm) and series resistance (Rs) and capacitance (Cm), were measured while clamping cells at −65 mV and applying voltage steps from −55 to −75 mV. The current-firing relationship was determined in current clamp with 1-s periods of injected current from 100 to 500 pA. The time course of viral gene expression experiments were carried out in organotypic hippocampal slice cultures prepared from postnatal day 5-7 Sprague-Dawley rats as described previously (Stoppini et al., 1991) .", "The time course of viral gene expression experiments were carried out in organotypic hippocampal slice cultures prepared from postnatal day 5-7 Sprague-Dawley rats as described previously (Stoppini et al., 1991) . Slices were infected after 7 days in vitro, and images were acquired on a two-photon microscope." ]

[ "Defining the connections among neurons is critical to our understanding of the structure and function of the nervous system. Recombinant viruses engineered to transmit across synapses provide a powerful approach for the dissection of neuronal circuitry in vivo. We recently demonstrated that recombinant vesicular stomatitis virus (VSV) can be endowed with anterograde or retrograde transsynaptic tracing ability by providing the virus with different glycoproteins.", "We recently demonstrated that recombinant vesicular stomatitis virus (VSV) can be endowed with anterograde or retrograde transsynaptic tracing ability by providing the virus with different glycoproteins. Here we extend the characterization of the transmission and gene expression of recombinant VSV (rVSV) with the rabies virus glycoprotein (RABV-G), and provide examples of its activity relative to the anterograde transsynaptic tracer form of rVSV. rVSV with RABV-G was found to drive strong expression of transgenes and to spread rapidly from neuron to neuron in only a retrograde manner.", "rVSV with RABV-G was found to drive strong expression of transgenes and to spread rapidly from neuron to neuron in only a retrograde manner. Depending upon how the RABV-G was delivered, VSV served as a polysynaptic or monosynaptic tracer, or was able to define projections through axonal uptake and retrograde transport. In animals co-infected with rVSV in its anterograde form, rVSV with RABV-G could be used to begin to characterize the similarities and differences in connections to different areas.", "In animals co-infected with rVSV in its anterograde form, rVSV with RABV-G could be used to begin to characterize the similarities and differences in connections to different areas. rVSV with RABV-G provides a flexible, rapid, and versatile tracing tool that complements the previously described VSV-based anterograde transsynaptic tracer. Text: Mapping neuronal connectivity in the central nervous system (CNS) of even simple organisms is a difficult task.", "Text: Mapping neuronal connectivity in the central nervous system (CNS) of even simple organisms is a difficult task. Recombinant viruses engineered to trace synaptic connections and express transgenes promise to enable higher-throughput mapping of connections among neurons than other methods, e.g., serial reconstruction from electron micrographs (Bock et al., 2011; Briggman et al., 2011) . The Pseudorabies (PRV) and Rabies viruses (RABV) have been the best characterized and most utilized circuit tracing viruses to date (Ugolini et al., 1989; Kelly and Strick, 2000) .", "The Pseudorabies (PRV) and Rabies viruses (RABV) have been the best characterized and most utilized circuit tracing viruses to date (Ugolini et al., 1989; Kelly and Strick, 2000) . RABV was recently modified by Wickersham and colleagues such that it can travel across only one synapse, allowing for a straightforward definition of monosynaptic connections (Wickersham et al., 2007b) . This strategy permitted the first unambiguous identification of retrogradely connected cells from an initially infected cell (\"starter cell\"), without the need for electrophysiology.", "This strategy permitted the first unambiguous identification of retrogradely connected cells from an initially infected cell (\"starter cell\"), without the need for electrophysiology. Moreover, the starter cell could be defined through the expression of a specific viral receptor that limited the initial infection. Recently, we created an anterograde monosynaptic virus that complements the previously available retrograde viral tracers (Beier et al., 2011) .", "Recently, we created an anterograde monosynaptic virus that complements the previously available retrograde viral tracers (Beier et al., 2011) . Vesicular stomatitis virus (VSV), a virus related to RABV, with its own glycoprotein (G) gene (VSV-G), or with a G from the unrelated lymphocytic choriomeningitis virus (LCMV-G), spreads in the anterograde direction across synapses. VSV can be used as a polysynaptic tracer that spreads across many synapses, owing to the fact that the normal, replicationcompetent form of the virus does not cause serious diseases in humans (Brandly and Hanson, 1957; Johnson et al., 1966; Brody et al., 1967) .", "VSV can be used as a polysynaptic tracer that spreads across many synapses, owing to the fact that the normal, replicationcompetent form of the virus does not cause serious diseases in humans (Brandly and Hanson, 1957; Johnson et al., 1966; Brody et al., 1967) . Whether the virus is a monosynaptic or polysynaptic tracer is determined by the method of delivery of the G gene ( Figure 1A) . Advantages of VSV are that it is well-characterized, is relatively simple in comparison to PRV, and it rapidly grows to high titer in tissue culture cells.", "Advantages of VSV are that it is well-characterized, is relatively simple in comparison to PRV, and it rapidly grows to high titer in tissue culture cells. It is also being developed as a vaccine vector, often using a G of another virus as the immunogen, as well as being developed as a cytocidal agent that will target tumor cells in humans (Balachandran and Barber, 2000; Stojdl et al., 2000 Stojdl et al., , 2003 . Previous studies of the anatomical patterns of transmission, as well as physiological recordings, have shown that the transmission of VSV and RABV among neurons is via synapses (Kelly and Strick, 2000; Wickersham et al., 2007b; Beier et al., 2011) .", "Previous studies of the anatomical patterns of transmission, as well as physiological recordings, have shown that the transmission of VSV and RABV among neurons is via synapses (Kelly and Strick, 2000; Wickersham et al., 2007b; Beier et al., 2011) . In addition, it has been shown that RABV, as well as lentiviruses with RABV-G in their envelope, travel retrogradely from an injection site (Mazarakis et al., 2001; Wickersham et al., 2007a) . We hypothesized that providing a recombinant VSV (rVSV) with the RABV-G would create a retrograde polysynaptic transsynaptic tracer without the biosafety concerns inherent to RABV.", "We hypothesized that providing a recombinant VSV (rVSV) with the RABV-G would create a retrograde polysynaptic transsynaptic tracer without the biosafety concerns inherent to RABV. Our initial characterization of rVSV with RABV-G showed that indeed FIGURE 1 | Synaptic tracing strategies using VSV. (A) Schematic illustrating the strategies for polysynaptic or monosynaptic retrograde or anterograde transsynaptic transmission of rVSV encoding GFP.", "(A) Schematic illustrating the strategies for polysynaptic or monosynaptic retrograde or anterograde transsynaptic transmission of rVSV encoding GFP. The initially infected cell is indicated by an asterisk. VSV encoding a glycoprotein (G) within its genome can spread polysynaptically. The direction of the spread depends on the identity of the glycoprotein.", "The direction of the spread depends on the identity of the glycoprotein. Infected neurons are shown in green. In some cases, the initially infected starter cell can be defined by the expression of an avian receptor, TVA (tagged with a red fluorescent protein).", "In some cases, the initially infected starter cell can be defined by the expression of an avian receptor, TVA (tagged with a red fluorescent protein). The TVA-expressing neurons can then be specifically infected by rVSV G with the EnvA/RABV-G (A/RG) glycoprotein (Wickersham et al., 2007b) on the virion surface [rVSV G(A/RG)]. These starter cells are then yellow, due to viral GFP and mCherry from TVA-mCherry expression.", "These starter cells are then yellow, due to viral GFP and mCherry from TVA-mCherry expression. For monosynaptic tracing, the G protein is expressed in trans in the TVA-expressing cell, and thus complements rVSV G to allow transmission in a specific direction. (B) Genomic diagrams of rVSV vectors.", "(B) Genomic diagrams of rVSV vectors. All VSVs contain four essential proteins: N, P, M, and L. Some viruses encode a G gene in their genome, which allows them to spread polysynaptically. rVSV vectors typically encode a transgene in the first position, while others carry an additional transgene in the G position.", "rVSV vectors typically encode a transgene in the first position, while others carry an additional transgene in the G position. (C) Morphological characterization of rVSV-infected neurons in several locations within the mouse brain. (i,ii) Caudate-putamen (CP) neurons at 4 dpi from an injection of the CP with rVSV(VSV-G) viruses encoding (i) CFP or (ii) Korange.", "(i,ii) Caudate-putamen (CP) neurons at 4 dpi from an injection of the CP with rVSV(VSV-G) viruses encoding (i) CFP or (ii) Korange. (iii) Labeled neurons of the CA1 region of the hippocampus are shown at 5 dpi following injection into the hippocampus of rVSV(VSV-G) encoding Venus. (iv,v) Cortical pyramidal neurons are shown following injection into the CP of rVSV(RABV-G) expressing (iv) GFP at 24 hpi, or (v) mCherry at 48 hpi.", "(iv,v) Cortical pyramidal neurons are shown following injection into the CP of rVSV(RABV-G) expressing (iv) GFP at 24 hpi, or (v) mCherry at 48 hpi. Inset in (iv) is a high magnification of the neuron in panel (iv), highlighting labeling of dendritic spines. (vi) Multiple viruses can be co-injected into the same animal.", "(vi) Multiple viruses can be co-injected into the same animal. Here, individual rVSV G(VSV-G) viruses encoding CFP, GFP , Venus, Korange, and mCherry were used to infect the cortex. Scale bars = 50 µm.", "Scale bars = 50 µm. February 2013 | Volume 7 | Article 11 | 2 it could be taken up as a retrograde tracer (Beier et al., 2011) . To determine if it could transmit among neurons following its replication in neurons, and to further analyze the transmission patterns of both the monosynaptic and polysynaptic forms of rVSV with RABV-G, we made injections into several CNS and peripheral locations.", "To determine if it could transmit among neurons following its replication in neurons, and to further analyze the transmission patterns of both the monosynaptic and polysynaptic forms of rVSV with RABV-G, we made injections into several CNS and peripheral locations. In addition, we performed co-infections of rVSV with RABV-G and the anterograde form of rVSV in order to exploit the differences in the directionality of transmission of these two viruses in mapping circuits. Schematics of viruses created and used throughout this study are shown in Figure 1 .", "Schematics of viruses created and used throughout this study are shown in Figure 1 . We created rVSV vector plasmids carrying different transgenes in either the first or fifth genomic positions ( Figure 1B) . After rescuing each virus, we tested the ability of each to express transgenes in different brain regions through intracranial injections ( Figure 1C ).", "After rescuing each virus, we tested the ability of each to express transgenes in different brain regions through intracranial injections ( Figure 1C ). All rVSV vectors drove robust fluorophore expression 1 or 2 days post-infection (hpi) ( Figure 1C ) (van den Pol et al., 2009) . In fact, by 12 hpi, labeling was sufficiently bright to image fine morphological details, such as dendritic spines ( Figure 1C ,iv).", "In fact, by 12 hpi, labeling was sufficiently bright to image fine morphological details, such as dendritic spines ( Figure 1C ,iv). To characterize the physiological properties of cells infected with rVSV, we tested a replication-competent rVSV encoding GFP, with RABV-G in the genome in place of VSV-G [hereafter designated rVSV(RABV-G)]. van den Pol et al.", "van den Pol et al. reported that hippocampal neurons infected with replication-incompetent (G-deleted or \" G\") rVSV were physiologically healthy at 12-14 hpi, but were less so by 1 day post-infection (dpi) (van den Pol et al., 2009) . Given the known toxicity of both VSV and RABV-G (Coulon et al., 1982) , we tested the physiology of cortical pyramidal neurons in the motor cortex (M1) infected with rVSV(RABV-G).", "Given the known toxicity of both VSV and RABV-G (Coulon et al., 1982) , we tested the physiology of cortical pyramidal neurons in the motor cortex (M1) infected with rVSV(RABV-G). Between 12 and 18 hpi, the membrane capacitance, input resistance, resting membrane potential, and current-to-action potential firing relationship were indistinguishable between infected and uninfected neurons (Figure 2) . However, by 2 dpi, electrophysiological properties were so abnormal in the infected cortical pyramidal cells that physiological measurements could not be made.", "However, by 2 dpi, electrophysiological properties were so abnormal in the infected cortical pyramidal cells that physiological measurements could not be made. The speed and strength of the expression of transgenes encoded by VSV depends upon the gene's genomic position (van den Pol et al., 2009; Beier et al., 2011) . Genes in the first position are expressed the most highly, with a decrease in the level of expression in positions more 3 within the viral plus strand.", "Genes in the first position are expressed the most highly, with a decrease in the level of expression in positions more 3 within the viral plus strand. When GFP was inserted into the first position of VSV, GFP fluorescence was first detectable at approximately 1 hpi in cultured cells (van den Pol et al., 2009) . In order to quantify the relative expression of a fluorescent protein in the first genomic position in neurons, rat hippocampal slices were infected with a replication-incompetent rVSV that expresses mCherry (rVSV G, Figures 1A,B) .", "In order to quantify the relative expression of a fluorescent protein in the first genomic position in neurons, rat hippocampal slices were infected with a replication-incompetent rVSV that expresses mCherry (rVSV G, Figures 1A,B) . This was a G virus which had the RABV-G supplied in trans during the preparation of the virus stock [referred to as rVSV G(RABV-G)]. Average fluorescence intensity of the infected cells was measured every hour over the course of 18 h. By 4 hpi at 37 • C, red fluorescence was clearly visible, and reached maximal levels by approximately 14 hpi (N = 3, Figure 3 ).", "Average fluorescence intensity of the infected cells was measured every hour over the course of 18 h. By 4 hpi at 37 • C, red fluorescence was clearly visible, and reached maximal levels by approximately 14 hpi (N = 3, Figure 3 ). Similar results were obtained with a virus encoding GFP in the first genomic position rather than mCherry (i.e., Figure 1B ) (N = 3). We previously demonstrated that rVSV(RABV-G) could be taken up retrogradely by neurons (Beier et al., 2011) , but these experiments did not distinguish between direct axonal uptake of the initial inoculum vs. retrograde transsynaptic transmission following viral replication.", "We previously demonstrated that rVSV(RABV-G) could be taken up retrogradely by neurons (Beier et al., 2011) , but these experiments did not distinguish between direct axonal uptake of the initial inoculum vs. retrograde transsynaptic transmission following viral replication. To distinguish between these two mechanisms and to extend the previous analyses, we conducted further experiments in the mammalian visual system (Figures 4A-G) . As visual cortex area 1 (V1) does not receive direct projections from retinal ganglion cells (RGCs), but rather receives secondary input from RGCs via the lateral geniculate nucleus (LGN), infection of RGCs from injection of V1 would demonstrate retrograde transmission from cells which supported at least one round of viral replication.", "As visual cortex area 1 (V1) does not receive direct projections from retinal ganglion cells (RGCs), but rather receives secondary input from RGCs via the lateral geniculate nucleus (LGN), infection of RGCs from injection of V1 would demonstrate retrograde transmission from cells which supported at least one round of viral replication. Following a V1 injection with rVSV(RABV-G), GFP-positive RGCs were observed in the retina by 3 dpi (N = 3; Figure 4G ). Importantly, viral labeling in the brain was restricted to primary and secondary projection areas, even at 7 dpi.", "Importantly, viral labeling in the brain was restricted to primary and secondary projection areas, even at 7 dpi. These included the LGN ( Figure 4D ) and the hypothalamus (Figure 4E) , two areas known to project directly to V1 (Kandel, 2000) . Selective labeling was observed in other areas, such as cortical areas surrounding V1 (Figure 4C) , which project directly to V1, and also in the superior colliculus (SC) stratum griseum centrale, which projects to the LGN ( Figure 4F) .", "Selective labeling was observed in other areas, such as cortical areas surrounding V1 (Figure 4C) , which project directly to V1, and also in the superior colliculus (SC) stratum griseum centrale, which projects to the LGN ( Figure 4F) . Labeling was also observed in the nucleus basalis, which projects to the cortex, as well as many components of the basal ganglia circuit, which provide input to the thalamus [such as the caudate-putamen (CP), globus pallidus (GP), and the subthalamic nucleus (STn)]. The amygdala, which projects to the hypothalamus, was also labeled.", "The amygdala, which projects to the hypothalamus, was also labeled. Consistent with a lack of widespread viral transmission, animals did not exhibit signs of disease at 7 dpi. These data show that rVSV(RABV-G) can spread in a retrograde direction from the injection site, but do not address whether the virus can spread exclusively in the retrograde direction.", "These data show that rVSV(RABV-G) can spread in a retrograde direction from the injection site, but do not address whether the virus can spread exclusively in the retrograde direction. Directional transsynaptic specificity can only be definitively addressed using a unidirectional circuit. We therefore turned to the primary motor cortex (M1) to CP connection, in which neurons project from the cortex to the CP, but not in the other direction ( Figure 4H ) (Beier et al., 2011) .", "We therefore turned to the primary motor cortex (M1) to CP connection, in which neurons project from the cortex to the CP, but not in the other direction ( Figure 4H ) (Beier et al., 2011) . Injections of rVSV(RABV-G) into M1 should not label neurons in the CP if the virus can only label cells across synapses in the retrograde direction. Indeed, at 2 dpi, areas directly projecting to the injection site, including the contralateral cortex, were labeled ( Figure 4I ).", "Indeed, at 2 dpi, areas directly projecting to the injection site, including the contralateral cortex, were labeled ( Figure 4I ). Only axons from cortical cells were observed in the CP, with no GFP-labeled cell bodies present in the CP (Figure 4J) , consistent with lack of anterograde transsynaptic spread. By 3 dpi, a small number of medium spiny neurons (MSNs) in the CP were observed, likely via secondary spread from initially infected thalamic or GP neurons (data not shown).", "By 3 dpi, a small number of medium spiny neurons (MSNs) in the CP were observed, likely via secondary spread from initially infected thalamic or GP neurons (data not shown). A particular advantage of retrograde viral tracers is the ability to label CNS neurons projecting to peripheral sites. This has been a powerful application of both RABV and PRV (Ugolini et al., 1989; Standish et al., 1994) .", "This has been a powerful application of both RABV and PRV (Ugolini et al., 1989; Standish et al., 1994) . To test if rVSV(RABV-G) could also perform this function, we examined the innervation of the dura surface by neurons of the trigeminal ganglion, a neuronal circuit thought to be involved in migraine headaches (Penfield and McNaughton, 1940; Mayberg et al., 1984) . These neurons have axons, but not canonical dendrites, and send projections into the spinal cord and brainstem.", "These neurons have axons, but not canonical dendrites, and send projections into the spinal cord and brainstem. Therefore, the only way trigeminal neurons could become labeled from viral application to the dura is through retrograde uptake of the virus. We applied rVSV(RABV-G) to the intact dura mater and analyzed the dura, trigeminal ganglion, and CNS for labeling ( Figure 4K ).", "We applied rVSV(RABV-G) to the intact dura mater and analyzed the dura, trigeminal ganglion, and CNS for labeling ( Figure 4K ). At the earliest time point examined, 3 dpi, we observed axons traveling along the dura, but little other evidence of infection ( Figure 4L) . No labeled neuronal cell bodies on the dura were observed, consistent with the lack of neurons on this surface.", "No labeled neuronal cell bodies on the dura were observed, consistent with the lack of neurons on this surface. In contrast, we did find labeled cell bodies in the trigeminal ganglion ( Figure 4M ). No infection was seen in the CNS, even at 4 dpi, consistent with the lack of inputs from the brain into the trigeminal ganglion (N = 4 animals).", "No infection was seen in the CNS, even at 4 dpi, consistent with the lack of inputs from the brain into the trigeminal ganglion (N = 4 animals). To further characterize patterns and kinetics of viral transmission and directional specificity of transsynaptic spread, injections of rVSV(RABV-G) were made into the CP ( Figure 5A ). In order to determine which cells were labeled by direct uptake of virus in the inoculum, a separate set of animals were injected into the CP with the replication-incompetent rVSV G(RABV-G) (N = 3 animals, analyzed 3 dpi).", "In order to determine which cells were labeled by direct uptake of virus in the inoculum, a separate set of animals were injected into the CP with the replication-incompetent rVSV G(RABV-G) (N = 3 animals, analyzed 3 dpi). Cells labeled by rVSV G(RABV-G) were observed in the CP, GP, substantia nigra (SN), thalamus, and layers 3 and 5 of the cortex, consistent with infection at the axon terminal and retrograde labeling of cell bodies of neurons known to project directly to the CP ( Figure 5C ) (Albin et al., 1995) . Areas labeled by CP injection are indicated in Figure 5B .", "Areas labeled by CP injection are indicated in Figure 5B . The patterns of spread for the replication-competent rVSV(RABV-G) were characterized over the course of 1-5 dpi ( Figures 5D-H) . During this interval, progressively more cells in infected regions were labeled by rVSV(RABV-G), including within the CP, nucleus basalis, cortex, and GP (listed in Figure 5B ).", "During this interval, progressively more cells in infected regions were labeled by rVSV(RABV-G), including within the CP, nucleus basalis, cortex, and GP (listed in Figure 5B ). In addition, more cortical cells were labeled in clusters near cortical pyramidal neurons, both ipsilateral and contralateral to the injected side, including neurogliaform cells (data not shown). These data are in contrast to those observed following infection with an anterograde transsynaptic tracing virus, such as rVSV with its own G gene, rVSV(VSV-G) (Figure 5B) .", "These data are in contrast to those observed following infection with an anterograde transsynaptic tracing virus, such as rVSV with its own G gene, rVSV(VSV-G) (Figure 5B) . At 3 dpi following rVSV(VSV-G) injection into the CP, the cerebral cortex was not labeled, but regions receiving projections from the CP, such as the STn, GP, and SN, were labeled (Beier et al., 2011) . In order to investigate other areas for evidence of cell-to-cell retrograde transsynaptic spread, the nucleus basalis was examined following infection of the CP with replication-competent rVSV(RABV-G).", "In order to investigate other areas for evidence of cell-to-cell retrograde transsynaptic spread, the nucleus basalis was examined following infection of the CP with replication-competent rVSV(RABV-G). The nucleus basalis was labeled by 2 dpi (Figures 5E-H) , consistent with at least a single transsynaptic jump, as this area does not directly project to the CP. The virus appeared to travel transsynaptically at the rate of roughly 1 synapse per day, as evidenced by the lack of labeled neurogliaform cells in the cortex, and lack of neurons in the nucleus basalis at 1 dpi, and label appearing in these cell types/areas at 2 dpi, as previously observed (Beier et al., 2011) .", "The virus appeared to travel transsynaptically at the rate of roughly 1 synapse per day, as evidenced by the lack of labeled neurogliaform cells in the cortex, and lack of neurons in the nucleus basalis at 1 dpi, and label appearing in these cell types/areas at 2 dpi, as previously observed (Beier et al., 2011) . Labeling remained well-restricted to the expected corticostriatal circuits at 5 dpi, suggesting that viral spread becomes less efficient after crossing one or two connections, consistent with injections into V1 (Figure 4) . While glial cells can be infected and were observed near the injection site (van den Pol et al., 2002; Chauhan et al., 2010) , infected glial cells away from the injection site generally were not observed.", "While glial cells can be infected and were observed near the injection site (van den Pol et al., 2002; Chauhan et al., 2010) , infected glial cells away from the injection site generally were not observed. One advantage of having both anterograde and retrograde forms of the same virus is that they can be used in parallel, or in tandem, to trace circuitry to and from a single or multiple sites of injection, with each virus having similar kinetics of spread and gene expression. In fact, if different fluorophores are used in different viruses, e.g., rVSV(VSV-G) and rVSV(RABV-G), then the viruses can be co-injected into the same site and their transmission can be traced independently ( Figure 6A ).", "In fact, if different fluorophores are used in different viruses, e.g., rVSV(VSV-G) and rVSV(RABV-G), then the viruses can be co-injected into the same site and their transmission can be traced independently ( Figure 6A ). This is most straightforward if there are no cells at the injection site that are initially infected by both viruses. Co-infected cells can be easily detected, as they would express both fluorescent proteins shortly after injection.", "Co-infected cells can be easily detected, as they would express both fluorescent proteins shortly after injection. In order to determine whether two viruses would allow simultaneous anterograde and retrograde transsynaptic tracing from a single injection site, a rVSV(VSV-G) expressing Venus and a rVSV(RABV-G) expressing mCherry were injected individually (Figures 6B-D) or co-injected (Figures 6E-G) into the motor cortex, and brains were examined 3 dpi. The pattern of labeling from the co-injected brains was equivalent to the patterns observed when each virus was injected individually: rVSV(VSV-G) was observed to infect neurons in the cortex, CP, and downstream nuclei, whereas the rVSV(RABV-G) was not observed to infect neurons in the CP, but rather in the thalamus and nucleus basalis (N = 4).", "The pattern of labeling from the co-injected brains was equivalent to the patterns observed when each virus was injected individually: rVSV(VSV-G) was observed to infect neurons in the cortex, CP, and downstream nuclei, whereas the rVSV(RABV-G) was not observed to infect neurons in the CP, but rather in the thalamus and nucleus basalis (N = 4). The initial co-infection rate is dependent upon The presence or absence of labeling is indicated by (+) and (−), respectively. The extent of labeling is indicated by the number of (+).", "The extent of labeling is indicated by the number of (+). Some animals were infected with G viruses to determine which areas were labeled by direct uptake of the virions, rather than by replication and transmission. These were sacrificed at 3 dpi.", "These were sacrificed at 3 dpi. (C) Parasaggital section of a brain infected with VSV[greek delta]G(RABV-G). The injection site is marked by a red arrow.", "The injection site is marked by a red arrow. Several areas that project directly to the CP were labeled due to direct uptake of the virions, including the cortex, thalamus, and GP (arrowheads), 3 dpi. the dose of the initial inocula.", "the dose of the initial inocula. When injecting 3 × 10 3 focus forming units (ffu) rVSV(VSV-G) and 3 × 10 4 ffu rVSV(RABV-G), no co-infection was observed at the injection site. Thus, co-infection of the same brain region, without co-infection of the same cells,\n\ndoes not alter the spreading behavior of either rVSV(VSV-G) or rVSV(RABV-G).", "Thus, co-infection of the same brain region, without co-infection of the same cells,\n\ndoes not alter the spreading behavior of either rVSV(VSV-G) or rVSV(RABV-G). One example of how this dual retrograde and anterograde transsynaptic tracing system can be used is to determine if three Frontiers in Neural Circuits February 2013 | Volume 7 | Article 11 | 8 distinct regions are connected and the directionality of any connections. For example, the anterograde transsynaptic virus can be injected into one region, the retrograde into another, and a third region can then be examined for evidence of labeling by either or both viruses (e.g., Figure 6H ).", "For example, the anterograde transsynaptic virus can be injected into one region, the retrograde into another, and a third region can then be examined for evidence of labeling by either or both viruses (e.g., Figure 6H ). To test this possibility, rVSV(VSV-G) was injected into the motor cortex, rVSV(RABV-G) was injected into the substantia nigra pars reticulara (SNr), and animals were sacrificed at 3 dpi. We observed that cells were singly labeled, either with Venus [rVSV(VSV-G)] or with mCherry [rVSV(RABV-G)], and were located largely in different regions of the CP (Figures 6I,J) (N = 3) .", "We observed that cells were singly labeled, either with Venus [rVSV(VSV-G)] or with mCherry [rVSV(RABV-G)], and were located largely in different regions of the CP (Figures 6I,J) (N = 3) . These results suggest that the anterograde connections from the cells infected with rVSV(VSV-G) in the M1 were with CP MSNs that did not project to the region of the SNr injected with rVSV(RABV-G) (N = 3 animals). In addition to polysynaptic tracing, VSV can be modified to trace circuits monosynaptically (Beier et al., 2011) .", "In addition to polysynaptic tracing, VSV can be modified to trace circuits monosynaptically (Beier et al., 2011) . With RABV, this was achieved in vivo by first infecting with an adeno-associated virus (AAV) expressing TVA, a receptor for an avian retrovirus, and RABV-G (Wall et al., 2010) . This was followed 3 weeks later by infection with a G RABV with an EnvA/RABV-G chimeric glycoprotein on the virion surface (Wickersham et al., 2007b) , which allowed infection specifically of the cells expressing TVA.", "This was followed 3 weeks later by infection with a G RABV with an EnvA/RABV-G chimeric glycoprotein on the virion surface (Wickersham et al., 2007b) , which allowed infection specifically of the cells expressing TVA. A similar strategy was used to test rVSV's ability to monosynaptically trace retrogradely connected neurons in vivo. Inputs to choline acetyltransferase (ChAT)-expressing neurons in the striatum were used for this test.", "Inputs to choline acetyltransferase (ChAT)-expressing neurons in the striatum were used for this test. These neurons primarily receive input from the cortex and the thalamus (Thomas et al., 2000; Bloomfield et al., 2007) (Figure 7A) . In order to mark this population, we crossed ChAT-Cre mice to Ai9 mice, which express tdTomato in cells with a Cre expression history (Madisen et al., 2010) .", "In order to mark this population, we crossed ChAT-Cre mice to Ai9 mice, which express tdTomato in cells with a Cre expression history (Madisen et al., 2010) . Six-week-old mice from this cross were injected in the CP with two AAV vectors: one expressing a Cre-conditional (\"floxed\") TVA-mCherry fusion protein, and another expressing a floxed RABV-G. Two weeks later, the mice were injected in the same coordinates with rVSV G with the EnvA/RABV-G chimeric glycoprotein on the virion surface [rVSV G(A/RG)] (Beier et al., 2011) . Cells successfully infected with these two AAV vectors could host infection by a rVSV and should be able to produce rVSV virions with RABV-G on the surface.", "Cells successfully infected with these two AAV vectors could host infection by a rVSV and should be able to produce rVSV virions with RABV-G on the surface. Such starter cells should also express tdTomato and GFP. If rVSV were to be produced, and if it were to transmit across the synapse retrogradely, cortical and thalamic neurons should be labeled by GFP.", "If rVSV were to be produced, and if it were to transmit across the synapse retrogradely, cortical and thalamic neurons should be labeled by GFP. Mice injected with these AAV and rVSV viruses were sacrificed 5 days after rVSV infection, and brains analyzed for fluorescence. As expected for starter cells, some neurons in the CP expressed both tdTomato and GFP (Figure 7B) .", "As expected for starter cells, some neurons in the CP expressed both tdTomato and GFP (Figure 7B) . Outside of the CP, small numbers of GFP+ neurons that were not mCherry+ were observed in the cortex (Figures 7C,D) and thalamus (Figure 7E) , consistent with retrograde spread. Control animals not expressing Cre, or not injected with AAV encoding RABV-G, did not label cells in the cortex or thalamus (N = 3 for both controls and experimental condition).", "Control animals not expressing Cre, or not injected with AAV encoding RABV-G, did not label cells in the cortex or thalamus (N = 3 for both controls and experimental condition). Here, we report on the use of rVSV as a retrograde transsynaptic tracer for CNS circuitry. VSV can be modified to encode the RABV-G protein in the viral genome, allowing the virus to replicate and transmit across multiple synaptically connected cells, i.e., as a polysynaptic tracer.", "VSV can be modified to encode the RABV-G protein in the viral genome, allowing the virus to replicate and transmit across multiple synaptically connected cells, i.e., as a polysynaptic tracer. Alternatively, if the virus has the G gene deleted from its genome and RABV-G is provided in trans, it behaves as a monosynaptic tracer (Beier et al., 2011) . Although it has been known for many years that RABV travels retrogradely among neurons (Astic et al., 1993; Ugolini, 1995; Kelly and Strick, 2003) , and pseudotyping lentiviruses with RABV-G is sufficient for axonal transport (Mazarakis et al., 2001) , the retrograde transmission specificity among neurons had not been clearly shown to be a property of the G protein itself, as it might have been due to other viral proteins in addition to, or instead of, the viral G protein.", "Although it has been known for many years that RABV travels retrogradely among neurons (Astic et al., 1993; Ugolini, 1995; Kelly and Strick, 2003) , and pseudotyping lentiviruses with RABV-G is sufficient for axonal transport (Mazarakis et al., 2001) , the retrograde transmission specificity among neurons had not been clearly shown to be a property of the G protein itself, as it might have been due to other viral proteins in addition to, or instead of, the viral G protein. Since native VSV does not have these retrograde transsynaptic properties (van den Pol et al., 2002; Beier et al., 2011) , and the only alteration to the VSV genome was the substitution of the VSV G gene with the G gene of RABV, it is clear that the RABV glycoprotein is responsible for retrograde direction of viral transmission across synapses, at least in the case of rVSV. The early onset of gene expression from VSV relative to RABV (one hour vs. multiple hours) makes it beneficial in experimental paradigms in which the experiment needs to be done within a narrow window of time, such as tissue slices and explants.", "The early onset of gene expression from VSV relative to RABV (one hour vs. multiple hours) makes it beneficial in experimental paradigms in which the experiment needs to be done within a narrow window of time, such as tissue slices and explants. In addition, more than one transgene can be encoded in the viral genome without the need of a 2A or IRES element. The use of the first position of the genome enhances the expression level of the transgene inserted at that location, since VSV (and RABV) express genes in a transcriptional gradient; therefore, the first gene is the most highly transcribed (Knipe, 2007) .", "The use of the first position of the genome enhances the expression level of the transgene inserted at that location, since VSV (and RABV) express genes in a transcriptional gradient; therefore, the first gene is the most highly transcribed (Knipe, 2007) . This leads to rational predictions of expression levels so that one can choose the position of insertion of a transgene, or transgenes, according to this gradient and the desired level of expression. The size of the viral capsid is apparently not rigid, allowing for the inclusion of genomes that are substantially larger than the native genome, unlike the rigid capacity for some other viral vectors, such as AAV Yan et al., 2000) .", "The size of the viral capsid is apparently not rigid, allowing for the inclusion of genomes that are substantially larger than the native genome, unlike the rigid capacity for some other viral vectors, such as AAV Yan et al., 2000) . The fact that VSV can be made to spread anterogradely (Beier et al., 2011) or retrogradely across synapses with the change of a single gene affords several advantages over viral tracers that heretofore have not shown such flexibility in the directionality of tracing. In addition to the obvious application of tracing anterograde connections, combinations can be made to exploit the different forms of the virus.", "In addition to the obvious application of tracing anterograde connections, combinations can be made to exploit the different forms of the virus. One example that employs the simultaneous infection with an anterograde and retrograde form of VSV is demonstrated in Figure 6 . This experiment was designed to address whether the anterograde projections from the cortex to the CP would label the same brain regions as were labeled by a retrograde virus injected into the SN.", "This experiment was designed to address whether the anterograde projections from the cortex to the CP would label the same brain regions as were labeled by a retrograde virus injected into the SN. Although a block of superinfection by the virus may preclude infection of the same cell with multiple rVSVs, adjacent cells could still become labeled by different viruses (Whitaker-Dowling et al., 1983) . The observed results could be due to a preferential labeling by the anterograde transsynaptic virus of indirect pathway MSNs in this experiment, which then synapse onto the GP, thereby reflecting a viral bias.", "The observed results could be due to a preferential labeling by the anterograde transsynaptic virus of indirect pathway MSNs in this experiment, which then synapse onto the GP, thereby reflecting a viral bias. Alternatively, it could indicate that the cortical neurons in the injected region largely do not label the MSNs that project to the area of the SN injected with the retrograde virus. One further possibility is that too little virus was used to observe co-labeling of a given region.", "One further possibility is that too little virus was used to observe co-labeling of a given region. However, given the density of infection (i.e., Figures 6I,J) , the latter possibility seems unlikely. Additionally, the spread of the polysynaptic rVSV(RABV-G) appears to attenuate with increasing numbers of synapses crossed, permitting an analysis of more restricted viral spread.", "Additionally, the spread of the polysynaptic rVSV(RABV-G) appears to attenuate with increasing numbers of synapses crossed, permitting an analysis of more restricted viral spread. This is quite fortuitous, as if spread were to continue, it would lead to widespread infection and lethality. In addition, reconstruction of connectivity would be more difficult.", "In addition, reconstruction of connectivity would be more difficult. This reduced efficiency appears to also hold for the monosynaptic form of VSV complemented with RABV-G, as the efficiency of transmission appeared lower than the comparable experiment with RABV (Watabe-Uchida et al., 2012) . This is likely due to viral attenuation when VSV-G is replaced with RABV-G.\n\nWe were attracted to the use of VSV as a viral tracer due to its long track record as a safe, replication-competent laboratory agent.", "This is likely due to viral attenuation when VSV-G is replaced with RABV-G.\n\nWe were attracted to the use of VSV as a viral tracer due to its long track record as a safe, replication-competent laboratory agent. Laboratory workers using VSV have not contracted any diseases, and natural VSV infections among human populations in Central America and the southwestern United States (Rodríguez, 2002) occur without evident pathology (Johnson et al., 1966; Brody et al., 1967) . VSV was thus an attractive candidate for its use as a polysynaptic tracer for CNS studies, which requires an ability to replicate through multiple transmission cycles.", "VSV was thus an attractive candidate for its use as a polysynaptic tracer for CNS studies, which requires an ability to replicate through multiple transmission cycles. Both replicationcompetent and incompetent forms of VSV are in use under Biosafety Level 2 containment. Replication-competent RABV is Biosafety Level 3, due to the fact that infection with replicationcompetent RABV is almost always fatal to humans and in mice when infected intracerebrally (Smith, 1981; Knipe, 2007) .", "Replication-competent RABV is Biosafety Level 3, due to the fact that infection with replicationcompetent RABV is almost always fatal to humans and in mice when infected intracerebrally (Smith, 1981; Knipe, 2007) . Differences in pathogenicity between VSV and RABV are likely due to the ability of RABV to evade the innate immune system, particularly interferon (Hangartner et al., 2006; Junt et al., 2007; Lyles and Rupprecht, 2007; Rieder and Conzelmann, 2009; Iannacone et al., 2010) . VSV infection efficiently triggers an interferon response, and it has not evolved a method of escape from this response, unlike RABV (Brzózka et al., 2006) .", "VSV infection efficiently triggers an interferon response, and it has not evolved a method of escape from this response, unlike RABV (Brzózka et al., 2006) . In fact, VSV is being pursued as a vaccine for other viruses, including RABV (Lichty et al., 2004; Publicover et al., 2004; Kapadia et al., 2005; Schwartz et al., 2007; Iyer et al., 2009; Geisbert and Feldmann, 2011) . VSV does not typically spread beyond the initially infected site in the periphery (Kramer et al., 1983; Vogel and Fertsch, 1987) .", "VSV does not typically spread beyond the initially infected site in the periphery (Kramer et al., 1983; Vogel and Fertsch, 1987) . This likely is the cause of the minor or absent symptoms in humans and animals infected in nature. Polysynaptic VSV vectors are thus predicted to be much safer than polysynaptic RABV vectors.", "Polysynaptic VSV vectors are thus predicted to be much safer than polysynaptic RABV vectors. We have tested this prediction by injecting a series of mice in the footpads and hind leg muscles with rVSV(RABV-G), with the result that no injected animals showed any evidence of morbidity or mortality (Beier, Goz et al., in preparation) . While safer for laboratory workers than RABV, the main drawback to using VSV is its rapid cellular toxicity (van den Pol et al., 2009; Beier et al., 2011) .", "While safer for laboratory workers than RABV, the main drawback to using VSV is its rapid cellular toxicity (van den Pol et al., 2009; Beier et al., 2011) . Toxicity is due to suppression of cellular transcription and a block in the export of cellular RNAs from the nucleus to the cytoplasm (Black and Lyles, 1992; Her et al., 1997; Ahmed and Lyles, 1998; Petersen et al., 2000; von Kobbe et al., 2000) , as well as inhibition of the translation of cellular mRNAs (Francoeur et al., 1987; Jayakar et al., 2000; Kopecky et al., Frontiers in Neural Circuits February 2013 | Volume 7 | Article 11 | 10 2001). VSV is much quicker to enact its gene expression program than is RABV, such that cells suffer the toxic effects more quickly than after RABV infection.", "VSV is much quicker to enact its gene expression program than is RABV, such that cells suffer the toxic effects more quickly than after RABV infection. One aspect of VSV that can be exploited in the future to ameliorate the speed of toxicity is the use of VSV mutants and variants. One such mutant is the M51R, which permitted us to conduct physiological analyses of pre-and post-synaptic cells (Beier et al., 2011) .", "One such mutant is the M51R, which permitted us to conduct physiological analyses of pre-and post-synaptic cells (Beier et al., 2011) . We are in the process of examining the transmission properties of this mutant in vivo, as well as the effects of other mutations or viral variants on prolonging the health of neurons after infection. rVSV vectors can be used to study the connectivity of neuronal circuitry.", "rVSV vectors can be used to study the connectivity of neuronal circuitry. In addition to combinations of replication-competent forms of VSV, the replication-incompetent, monosynaptic forms of the virus can be easily combined, without the need to change viruses (Beier et al., 2011) . This allows a straightforward way to study both the projections into, and out from, a genetically defined cell population.", "This allows a straightforward way to study both the projections into, and out from, a genetically defined cell population. This can be done with the same viral genome, with the only change needed being the glycoprotein, for the selection of the direction of transmission. This flexibility of VSV makes it a powerful, multi-application vector for studying connectivity in the CNS.", "This flexibility of VSV makes it a powerful, multi-application vector for studying connectivity in the CNS. All rVSV clones were cloned from the rVSV G backbone (Chandran et al., 2005) . mCherry, Kusabira orange, Venus, and CFP were cloned into the first (GFP) position using XhoI and MscI sites, and VSV-G (a gift from Richard Mulligan, Harvard Medical School, Boston, MA) and RABV-G (a gift from Ed Callaway, Salk Institute, San Diego, CA) were cloned into the fifth (G) position using the MluI and NotI restriction sites.", "mCherry, Kusabira orange, Venus, and CFP were cloned into the first (GFP) position using XhoI and MscI sites, and VSV-G (a gift from Richard Mulligan, Harvard Medical School, Boston, MA) and RABV-G (a gift from Ed Callaway, Salk Institute, San Diego, CA) were cloned into the fifth (G) position using the MluI and NotI restriction sites. Genes for fluorescent proteins were obtained from Clontech. Viruses were rescued as previously described (Whelan et al., 1995) .", "Viruses were rescued as previously described (Whelan et al., 1995) . At 95% confluency, eight 10 cm plates of BSR cells were infected at an MOI of 0.01. Viral supernatants were collected at 24-h time intervals and ultracentrifuged at 21,000 RPM using a SW28 rotor and resuspended in 0.2% of the original volume.", "Viral supernatants were collected at 24-h time intervals and ultracentrifuged at 21,000 RPM using a SW28 rotor and resuspended in 0.2% of the original volume. For titering, concentrated viral stocks were applied in a dilution series to 100% confluent BSR cells and plates were examined at 12 hpi. Viral stocks were stored at −80 • C.\n\nFor G viruses, 293T cells were transfected with PEI (Ehrhardt et al., 2006) at 70% confluency on 10 cm dishes with 5 µg of pCAG-RABV-G. Twenty-four hours post-infection, the cells were infected at an MOI of 0.01 with rVSV G expressing either GFP or mCherry.", "Viral stocks were stored at −80 • C.\n\nFor G viruses, 293T cells were transfected with PEI (Ehrhardt et al., 2006) at 70% confluency on 10 cm dishes with 5 µg of pCAG-RABV-G. Twenty-four hours post-infection, the cells were infected at an MOI of 0.01 with rVSV G expressing either GFP or mCherry. Viral supernatants were collected for the subsequent 4 days at 24 h intervals. Virus preparations are now available from the Salk GT3 viral core ( All plasmids are available from Addgene (\n\nAAV-FLEx-RABV-G and AAV-FLEx-TVA-mCherry plasmids originated from the Lab of Naoshige Uchida (Watabe-Uchida et al., 2012) , and virus stocks were generous gifts from Brad Lowell, Harvard Medical School.", "Virus preparations are now available from the Salk GT3 viral core ( All plasmids are available from Addgene (\n\nAAV-FLEx-RABV-G and AAV-FLEx-TVA-mCherry plasmids originated from the Lab of Naoshige Uchida (Watabe-Uchida et al., 2012) , and virus stocks were generous gifts from Brad Lowell, Harvard Medical School. ChAT-Cre (B6;129S6-Chat tm1(cre)Lowl /J) and Ai9 (B6.Cg-Gt(ROSA)26Sor<tm9(CAG-tdTomato)Hze>/J) mice were obtained from the Jackson Laboratory (Madisen et al., 2010) . Eight-week-old CD-1 mice were injected using pulled capillary microdispensers (Drummond Scientific, Cat.", "Eight-week-old CD-1 mice were injected using pulled capillary microdispensers (Drummond Scientific, Cat. No: 5-000-2005) , using coordinates from The Mouse Brain in Stereotaxic Coordinates (Franklin and Paxinos, 1997) . Injection coordinates (in mm) used were: For multi-color analysis (Figures 1C,D) , 3 × 10 9 ffu/mL rVSV was injected into various regions.", "Injection coordinates (in mm) used were: For multi-color analysis (Figures 1C,D) , 3 × 10 9 ffu/mL rVSV was injected into various regions. For CP injections, 100 nL of rVSV(RABV-G) or rVSV(VSV-G) at 3 × 10 7 ffu/mL was injected at a rate of 100 nL/min. For the replication-incompetent viruses, 100 nL of 1 × 10 7 ffu/mL rVSV G(RABV-G) or rVSV G (VSV-G) was injected.", "For the replication-incompetent viruses, 100 nL of 1 × 10 7 ffu/mL rVSV G(RABV-G) or rVSV G (VSV-G) was injected. In the motor cortex, 100 nL of 1 × 10 7 ffu/mL rVSV(RABV-G) was injected, and mice harvested 2 dpi. For V1 injections, 100 nL of 3 × 10 10 ffu/mL rVSV(RABV-G) was injected, and mice were examined 3 or 7 dpi.", "For V1 injections, 100 nL of 3 × 10 10 ffu/mL rVSV(RABV-G) was injected, and mice were examined 3 or 7 dpi. For infections of the dura mater, 1 µL of 3 × 10 10 ffu/mL rVSV(RABV-G) was applied to the surface of the dura. The virus was allowed to absorb, and the surface was subsequently covered in bone wax, and the wound sutured.", "The virus was allowed to absorb, and the surface was subsequently covered in bone wax, and the wound sutured. For co-injections of virus into the same animal, 100 nL of a combination of 3 × 10 7 ffu/mL rVSV(VSV-G) and 3 × 10 8 ffu/mL rVSV(RABV-G) were co-injected into the motor cortex, and brains examined 3 dpi. For injections of the viruses into different regions, 100 nL of 3 × 10 7 ffu/mL rVSV(VSV-G) was injected into M1, and 100 nL of 3 × 10 8 ffu/mL rVSV(RABV-G) into the SNr, and brains examined 3 dpi.", "For injections of the viruses into different regions, 100 nL of 3 × 10 7 ffu/mL rVSV(VSV-G) was injected into M1, and 100 nL of 3 × 10 8 ffu/mL rVSV(RABV-G) into the SNr, and brains examined 3 dpi. A lower titer of rVSV(VSV-G) was used, as rVSV(RABV-G) is attenuated. All mouse work was conducted in biosafety containment level 2 conditions and was approved by the Longwood Medical Area Institutional Animal Care and Use Committee.", "All mouse work was conducted in biosafety containment level 2 conditions and was approved by the Longwood Medical Area Institutional Animal Care and Use Committee. Recordings were made from cortical pyramidal neurons in slices taken from postnatal day 12-18 mice, inoculated in the CP 12-18 h prior with rVSV(RABV-G). Coronal slices (300 µm thick) were cut in ice-cold external solution containing (in mM): 110 choline, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 2.5 KCl, 7 MgCl 2 , 0.5 CaCl 2 , 25 glucose, 11.6 Na-ascorbate, and 3.1 Na-pyruvate, bubbled with 95% O 2 and 5% CO 2 .", "Coronal slices (300 µm thick) were cut in ice-cold external solution containing (in mM): 110 choline, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 2.5 KCl, 7 MgCl 2 , 0.5 CaCl 2 , 25 glucose, 11.6 Na-ascorbate, and 3.1 Na-pyruvate, bubbled with 95% O 2 and 5% CO 2 . Slices were then transferred to artificial cerebrospinal fluid (ACSF) containing (in mM): 127 NaCl, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 2.5 KCl, 1 MgCl 2 , 2 CaCl 2 , and 25 glucose, bubbled with 95% O 2 and 5% CO 2 . After an incubation period Frontiers in Neural Circuits February 2013 | Volume 7 | Article 11 | 11 of 30-40 min at 34 • C, slices were stored at room temperature.", "After an incubation period Frontiers in Neural Circuits February 2013 | Volume 7 | Article 11 | 11 of 30-40 min at 34 • C, slices were stored at room temperature. All experiments were conducted at room temperature (25 • C). In all experiments, 50 µM picrotoxin, 10 µM 2,3-Dioxo-6-nitro-1, 2, 3, 4 -tetrahydrobenzo [f]quinoxaline -7-sulfonamide (NBQX), and 10 µM 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) were present in the ACSF to block GABAA/C, AMPA, and NMDA receptor-mediated transmission, respectively.", "In all experiments, 50 µM picrotoxin, 10 µM 2,3-Dioxo-6-nitro-1, 2, 3, 4 -tetrahydrobenzo [f]quinoxaline -7-sulfonamide (NBQX), and 10 µM 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) were present in the ACSF to block GABAA/C, AMPA, and NMDA receptor-mediated transmission, respectively. All chemicals were from Sigma or Tocris. Whole-cell recordings were obtained from infected and uninfected deep layer cortical pyramidal neurons identified with video-IR/DIC and GFP fluorescence was detected using epifluorescence illumination.", "Whole-cell recordings were obtained from infected and uninfected deep layer cortical pyramidal neurons identified with video-IR/DIC and GFP fluorescence was detected using epifluorescence illumination. With the deep layers of the cortex, 2-photon laser scanning microscopy (2PLSM) was used to confirm the cell types based on morphology. Deep layer pyramidal neurons had large cell bodies, classic pyramidal shape and dendritic spines.", "Deep layer pyramidal neurons had large cell bodies, classic pyramidal shape and dendritic spines. Glass electrodes (2-4 M ) were filled with internal solution containing (in mM): 135 KMeSO 4 , 5 KCl, 5 HEPES, 4 MgATP, 0.3 NaGTP, 10 Na 2 HPO 4 , 1 EGTA, and 0.01 Alexa Fluor-594 (to image neuronal morphology) adjusted to pH 7.4 with KOH. Current and voltage recordings were made at room temperature using a AxoPatch 200B or a Multiclamp 700B amplifier.", "Current and voltage recordings were made at room temperature using a AxoPatch 200B or a Multiclamp 700B amplifier. Data was filtered at 5 kHz and digitized at 10 kHz. Imaging and physiology data were acquired and analyzed as described previously (Carter and Sabatini, 2004) .", "Imaging and physiology data were acquired and analyzed as described previously (Carter and Sabatini, 2004) . Resting membrane potential was determined by the average of three 5-s sweeps with no injected current. Passive properties of the cell, membrane (Rm) and series resistance (Rs) and capacitance (Cm), were measured while clamping cells at −65 mV and applying voltage steps from −55 to −75 mV.", "Passive properties of the cell, membrane (Rm) and series resistance (Rs) and capacitance (Cm), were measured while clamping cells at −65 mV and applying voltage steps from −55 to −75 mV. The current-firing relationship was determined in current clamp with 1-s periods of injected current from 100 to 500 pA. The time course of viral gene expression experiments were carried out in organotypic hippocampal slice cultures prepared from postnatal day 5-7 Sprague-Dawley rats as described previously (Stoppini et al., 1991) .", "The time course of viral gene expression experiments were carried out in organotypic hippocampal slice cultures prepared from postnatal day 5-7 Sprague-Dawley rats as described previously (Stoppini et al., 1991) . Slices were infected after 7 days in vitro, and images were acquired on a two-photon microscope." ]

[ "Defining the connections among neurons is critical to our understanding of the structure and function of the nervous system. Recombinant viruses engineered to transmit across synapses provide a powerful approach for the dissection of neuronal circuitry in vivo. We recently demonstrated that recombinant vesicular stomatitis virus (VSV) can be endowed with anterograde or retrograde transsynaptic tracing ability by providing the virus with different glycoproteins.", "We recently demonstrated that recombinant vesicular stomatitis virus (VSV) can be endowed with anterograde or retrograde transsynaptic tracing ability by providing the virus with different glycoproteins. Here we extend the characterization of the transmission and gene expression of recombinant VSV (rVSV) with the rabies virus glycoprotein (RABV-G), and provide examples of its activity relative to the anterograde transsynaptic tracer form of rVSV. rVSV with RABV-G was found to drive strong expression of transgenes and to spread rapidly from neuron to neuron in only a retrograde manner.", "rVSV with RABV-G was found to drive strong expression of transgenes and to spread rapidly from neuron to neuron in only a retrograde manner. Depending upon how the RABV-G was delivered, VSV served as a polysynaptic or monosynaptic tracer, or was able to define projections through axonal uptake and retrograde transport. In animals co-infected with rVSV in its anterograde form, rVSV with RABV-G could be used to begin to characterize the similarities and differences in connections to different areas.", "In animals co-infected with rVSV in its anterograde form, rVSV with RABV-G could be used to begin to characterize the similarities and differences in connections to different areas. rVSV with RABV-G provides a flexible, rapid, and versatile tracing tool that complements the previously described VSV-based anterograde transsynaptic tracer. Text: Mapping neuronal connectivity in the central nervous system (CNS) of even simple organisms is a difficult task.", "Text: Mapping neuronal connectivity in the central nervous system (CNS) of even simple organisms is a difficult task. Recombinant viruses engineered to trace synaptic connections and express transgenes promise to enable higher-throughput mapping of connections among neurons than other methods, e.g., serial reconstruction from electron micrographs (Bock et al., 2011; Briggman et al., 2011) . The Pseudorabies (PRV) and Rabies viruses (RABV) have been the best characterized and most utilized circuit tracing viruses to date (Ugolini et al., 1989; Kelly and Strick, 2000) .", "The Pseudorabies (PRV) and Rabies viruses (RABV) have been the best characterized and most utilized circuit tracing viruses to date (Ugolini et al., 1989; Kelly and Strick, 2000) . RABV was recently modified by Wickersham and colleagues such that it can travel across only one synapse, allowing for a straightforward definition of monosynaptic connections (Wickersham et al., 2007b) . This strategy permitted the first unambiguous identification of retrogradely connected cells from an initially infected cell (\"starter cell\"), without the need for electrophysiology.", "This strategy permitted the first unambiguous identification of retrogradely connected cells from an initially infected cell (\"starter cell\"), without the need for electrophysiology. Moreover, the starter cell could be defined through the expression of a specific viral receptor that limited the initial infection. Recently, we created an anterograde monosynaptic virus that complements the previously available retrograde viral tracers (Beier et al., 2011) .", "Recently, we created an anterograde monosynaptic virus that complements the previously available retrograde viral tracers (Beier et al., 2011) . Vesicular stomatitis virus (VSV), a virus related to RABV, with its own glycoprotein (G) gene (VSV-G), or with a G from the unrelated lymphocytic choriomeningitis virus (LCMV-G), spreads in the anterograde direction across synapses. VSV can be used as a polysynaptic tracer that spreads across many synapses, owing to the fact that the normal, replicationcompetent form of the virus does not cause serious diseases in humans (Brandly and Hanson, 1957; Johnson et al., 1966; Brody et al., 1967) .", "VSV can be used as a polysynaptic tracer that spreads across many synapses, owing to the fact that the normal, replicationcompetent form of the virus does not cause serious diseases in humans (Brandly and Hanson, 1957; Johnson et al., 1966; Brody et al., 1967) . Whether the virus is a monosynaptic or polysynaptic tracer is determined by the method of delivery of the G gene ( Figure 1A) . Advantages of VSV are that it is well-characterized, is relatively simple in comparison to PRV, and it rapidly grows to high titer in tissue culture cells.", "Advantages of VSV are that it is well-characterized, is relatively simple in comparison to PRV, and it rapidly grows to high titer in tissue culture cells. It is also being developed as a vaccine vector, often using a G of another virus as the immunogen, as well as being developed as a cytocidal agent that will target tumor cells in humans (Balachandran and Barber, 2000; Stojdl et al., 2000 Stojdl et al., , 2003 . Previous studies of the anatomical patterns of transmission, as well as physiological recordings, have shown that the transmission of VSV and RABV among neurons is via synapses (Kelly and Strick, 2000; Wickersham et al., 2007b; Beier et al., 2011) .", "Previous studies of the anatomical patterns of transmission, as well as physiological recordings, have shown that the transmission of VSV and RABV among neurons is via synapses (Kelly and Strick, 2000; Wickersham et al., 2007b; Beier et al., 2011) . In addition, it has been shown that RABV, as well as lentiviruses with RABV-G in their envelope, travel retrogradely from an injection site (Mazarakis et al., 2001; Wickersham et al., 2007a) . We hypothesized that providing a recombinant VSV (rVSV) with the RABV-G would create a retrograde polysynaptic transsynaptic tracer without the biosafety concerns inherent to RABV.", "We hypothesized that providing a recombinant VSV (rVSV) with the RABV-G would create a retrograde polysynaptic transsynaptic tracer without the biosafety concerns inherent to RABV. Our initial characterization of rVSV with RABV-G showed that indeed FIGURE 1 | Synaptic tracing strategies using VSV. (A) Schematic illustrating the strategies for polysynaptic or monosynaptic retrograde or anterograde transsynaptic transmission of rVSV encoding GFP.", "(A) Schematic illustrating the strategies for polysynaptic or monosynaptic retrograde or anterograde transsynaptic transmission of rVSV encoding GFP. The initially infected cell is indicated by an asterisk. VSV encoding a glycoprotein (G) within its genome can spread polysynaptically. The direction of the spread depends on the identity of the glycoprotein.", "The direction of the spread depends on the identity of the glycoprotein. Infected neurons are shown in green. In some cases, the initially infected starter cell can be defined by the expression of an avian receptor, TVA (tagged with a red fluorescent protein).", "In some cases, the initially infected starter cell can be defined by the expression of an avian receptor, TVA (tagged with a red fluorescent protein). The TVA-expressing neurons can then be specifically infected by rVSV G with the EnvA/RABV-G (A/RG) glycoprotein (Wickersham et al., 2007b) on the virion surface [rVSV G(A/RG)]. These starter cells are then yellow, due to viral GFP and mCherry from TVA-mCherry expression.", "These starter cells are then yellow, due to viral GFP and mCherry from TVA-mCherry expression. For monosynaptic tracing, the G protein is expressed in trans in the TVA-expressing cell, and thus complements rVSV G to allow transmission in a specific direction. (B) Genomic diagrams of rVSV vectors.", "(B) Genomic diagrams of rVSV vectors. All VSVs contain four essential proteins: N, P, M, and L. Some viruses encode a G gene in their genome, which allows them to spread polysynaptically. rVSV vectors typically encode a transgene in the first position, while others carry an additional transgene in the G position.", "rVSV vectors typically encode a transgene in the first position, while others carry an additional transgene in the G position. (C) Morphological characterization of rVSV-infected neurons in several locations within the mouse brain. (i,ii) Caudate-putamen (CP) neurons at 4 dpi from an injection of the CP with rVSV(VSV-G) viruses encoding (i) CFP or (ii) Korange.", "(i,ii) Caudate-putamen (CP) neurons at 4 dpi from an injection of the CP with rVSV(VSV-G) viruses encoding (i) CFP or (ii) Korange. (iii) Labeled neurons of the CA1 region of the hippocampus are shown at 5 dpi following injection into the hippocampus of rVSV(VSV-G) encoding Venus. (iv,v) Cortical pyramidal neurons are shown following injection into the CP of rVSV(RABV-G) expressing (iv) GFP at 24 hpi, or (v) mCherry at 48 hpi.", "(iv,v) Cortical pyramidal neurons are shown following injection into the CP of rVSV(RABV-G) expressing (iv) GFP at 24 hpi, or (v) mCherry at 48 hpi. Inset in (iv) is a high magnification of the neuron in panel (iv), highlighting labeling of dendritic spines. (vi) Multiple viruses can be co-injected into the same animal.", "(vi) Multiple viruses can be co-injected into the same animal. Here, individual rVSV G(VSV-G) viruses encoding CFP, GFP , Venus, Korange, and mCherry were used to infect the cortex. Scale bars = 50 µm.", "Scale bars = 50 µm. February 2013 | Volume 7 | Article 11 | 2 it could be taken up as a retrograde tracer (Beier et al., 2011) . To determine if it could transmit among neurons following its replication in neurons, and to further analyze the transmission patterns of both the monosynaptic and polysynaptic forms of rVSV with RABV-G, we made injections into several CNS and peripheral locations.", "To determine if it could transmit among neurons following its replication in neurons, and to further analyze the transmission patterns of both the monosynaptic and polysynaptic forms of rVSV with RABV-G, we made injections into several CNS and peripheral locations. In addition, we performed co-infections of rVSV with RABV-G and the anterograde form of rVSV in order to exploit the differences in the directionality of transmission of these two viruses in mapping circuits. Schematics of viruses created and used throughout this study are shown in Figure 1 .", "Schematics of viruses created and used throughout this study are shown in Figure 1 . We created rVSV vector plasmids carrying different transgenes in either the first or fifth genomic positions ( Figure 1B) . After rescuing each virus, we tested the ability of each to express transgenes in different brain regions through intracranial injections ( Figure 1C ).", "After rescuing each virus, we tested the ability of each to express transgenes in different brain regions through intracranial injections ( Figure 1C ). All rVSV vectors drove robust fluorophore expression 1 or 2 days post-infection (hpi) ( Figure 1C ) (van den Pol et al., 2009) . In fact, by 12 hpi, labeling was sufficiently bright to image fine morphological details, such as dendritic spines ( Figure 1C ,iv).", "In fact, by 12 hpi, labeling was sufficiently bright to image fine morphological details, such as dendritic spines ( Figure 1C ,iv). To characterize the physiological properties of cells infected with rVSV, we tested a replication-competent rVSV encoding GFP, with RABV-G in the genome in place of VSV-G [hereafter designated rVSV(RABV-G)]. van den Pol et al.", "van den Pol et al. reported that hippocampal neurons infected with replication-incompetent (G-deleted or \" G\") rVSV were physiologically healthy at 12-14 hpi, but were less so by 1 day post-infection (dpi) (van den Pol et al., 2009) . Given the known toxicity of both VSV and RABV-G (Coulon et al., 1982) , we tested the physiology of cortical pyramidal neurons in the motor cortex (M1) infected with rVSV(RABV-G).", "Given the known toxicity of both VSV and RABV-G (Coulon et al., 1982) , we tested the physiology of cortical pyramidal neurons in the motor cortex (M1) infected with rVSV(RABV-G). Between 12 and 18 hpi, the membrane capacitance, input resistance, resting membrane potential, and current-to-action potential firing relationship were indistinguishable between infected and uninfected neurons (Figure 2) . However, by 2 dpi, electrophysiological properties were so abnormal in the infected cortical pyramidal cells that physiological measurements could not be made.", "However, by 2 dpi, electrophysiological properties were so abnormal in the infected cortical pyramidal cells that physiological measurements could not be made. The speed and strength of the expression of transgenes encoded by VSV depends upon the gene's genomic position (van den Pol et al., 2009; Beier et al., 2011) . Genes in the first position are expressed the most highly, with a decrease in the level of expression in positions more 3 within the viral plus strand.", "Genes in the first position are expressed the most highly, with a decrease in the level of expression in positions more 3 within the viral plus strand. When GFP was inserted into the first position of VSV, GFP fluorescence was first detectable at approximately 1 hpi in cultured cells (van den Pol et al., 2009) . In order to quantify the relative expression of a fluorescent protein in the first genomic position in neurons, rat hippocampal slices were infected with a replication-incompetent rVSV that expresses mCherry (rVSV G, Figures 1A,B) .", "In order to quantify the relative expression of a fluorescent protein in the first genomic position in neurons, rat hippocampal slices were infected with a replication-incompetent rVSV that expresses mCherry (rVSV G, Figures 1A,B) . This was a G virus which had the RABV-G supplied in trans during the preparation of the virus stock [referred to as rVSV G(RABV-G)]. Average fluorescence intensity of the infected cells was measured every hour over the course of 18 h. By 4 hpi at 37 • C, red fluorescence was clearly visible, and reached maximal levels by approximately 14 hpi (N = 3, Figure 3 ).", "Average fluorescence intensity of the infected cells was measured every hour over the course of 18 h. By 4 hpi at 37 • C, red fluorescence was clearly visible, and reached maximal levels by approximately 14 hpi (N = 3, Figure 3 ). Similar results were obtained with a virus encoding GFP in the first genomic position rather than mCherry (i.e., Figure 1B ) (N = 3). We previously demonstrated that rVSV(RABV-G) could be taken up retrogradely by neurons (Beier et al., 2011) , but these experiments did not distinguish between direct axonal uptake of the initial inoculum vs. retrograde transsynaptic transmission following viral replication.", "We previously demonstrated that rVSV(RABV-G) could be taken up retrogradely by neurons (Beier et al., 2011) , but these experiments did not distinguish between direct axonal uptake of the initial inoculum vs. retrograde transsynaptic transmission following viral replication. To distinguish between these two mechanisms and to extend the previous analyses, we conducted further experiments in the mammalian visual system (Figures 4A-G) . As visual cortex area 1 (V1) does not receive direct projections from retinal ganglion cells (RGCs), but rather receives secondary input from RGCs via the lateral geniculate nucleus (LGN), infection of RGCs from injection of V1 would demonstrate retrograde transmission from cells which supported at least one round of viral replication.", "As visual cortex area 1 (V1) does not receive direct projections from retinal ganglion cells (RGCs), but rather receives secondary input from RGCs via the lateral geniculate nucleus (LGN), infection of RGCs from injection of V1 would demonstrate retrograde transmission from cells which supported at least one round of viral replication. Following a V1 injection with rVSV(RABV-G), GFP-positive RGCs were observed in the retina by 3 dpi (N = 3; Figure 4G ). Importantly, viral labeling in the brain was restricted to primary and secondary projection areas, even at 7 dpi.", "Importantly, viral labeling in the brain was restricted to primary and secondary projection areas, even at 7 dpi. These included the LGN ( Figure 4D ) and the hypothalamus (Figure 4E) , two areas known to project directly to V1 (Kandel, 2000) . Selective labeling was observed in other areas, such as cortical areas surrounding V1 (Figure 4C) , which project directly to V1, and also in the superior colliculus (SC) stratum griseum centrale, which projects to the LGN ( Figure 4F) .", "Selective labeling was observed in other areas, such as cortical areas surrounding V1 (Figure 4C) , which project directly to V1, and also in the superior colliculus (SC) stratum griseum centrale, which projects to the LGN ( Figure 4F) . Labeling was also observed in the nucleus basalis, which projects to the cortex, as well as many components of the basal ganglia circuit, which provide input to the thalamus [such as the caudate-putamen (CP), globus pallidus (GP), and the subthalamic nucleus (STn)]. The amygdala, which projects to the hypothalamus, was also labeled.", "The amygdala, which projects to the hypothalamus, was also labeled. Consistent with a lack of widespread viral transmission, animals did not exhibit signs of disease at 7 dpi. These data show that rVSV(RABV-G) can spread in a retrograde direction from the injection site, but do not address whether the virus can spread exclusively in the retrograde direction.", "These data show that rVSV(RABV-G) can spread in a retrograde direction from the injection site, but do not address whether the virus can spread exclusively in the retrograde direction. Directional transsynaptic specificity can only be definitively addressed using a unidirectional circuit. We therefore turned to the primary motor cortex (M1) to CP connection, in which neurons project from the cortex to the CP, but not in the other direction ( Figure 4H ) (Beier et al., 2011) .", "We therefore turned to the primary motor cortex (M1) to CP connection, in which neurons project from the cortex to the CP, but not in the other direction ( Figure 4H ) (Beier et al., 2011) . Injections of rVSV(RABV-G) into M1 should not label neurons in the CP if the virus can only label cells across synapses in the retrograde direction. Indeed, at 2 dpi, areas directly projecting to the injection site, including the contralateral cortex, were labeled ( Figure 4I ).", "Indeed, at 2 dpi, areas directly projecting to the injection site, including the contralateral cortex, were labeled ( Figure 4I ). Only axons from cortical cells were observed in the CP, with no GFP-labeled cell bodies present in the CP (Figure 4J) , consistent with lack of anterograde transsynaptic spread. By 3 dpi, a small number of medium spiny neurons (MSNs) in the CP were observed, likely via secondary spread from initially infected thalamic or GP neurons (data not shown).", "By 3 dpi, a small number of medium spiny neurons (MSNs) in the CP were observed, likely via secondary spread from initially infected thalamic or GP neurons (data not shown). A particular advantage of retrograde viral tracers is the ability to label CNS neurons projecting to peripheral sites. This has been a powerful application of both RABV and PRV (Ugolini et al., 1989; Standish et al., 1994) .", "This has been a powerful application of both RABV and PRV (Ugolini et al., 1989; Standish et al., 1994) . To test if rVSV(RABV-G) could also perform this function, we examined the innervation of the dura surface by neurons of the trigeminal ganglion, a neuronal circuit thought to be involved in migraine headaches (Penfield and McNaughton, 1940; Mayberg et al., 1984) . These neurons have axons, but not canonical dendrites, and send projections into the spinal cord and brainstem.", "These neurons have axons, but not canonical dendrites, and send projections into the spinal cord and brainstem. Therefore, the only way trigeminal neurons could become labeled from viral application to the dura is through retrograde uptake of the virus. We applied rVSV(RABV-G) to the intact dura mater and analyzed the dura, trigeminal ganglion, and CNS for labeling ( Figure 4K ).", "We applied rVSV(RABV-G) to the intact dura mater and analyzed the dura, trigeminal ganglion, and CNS for labeling ( Figure 4K ). At the earliest time point examined, 3 dpi, we observed axons traveling along the dura, but little other evidence of infection ( Figure 4L) . No labeled neuronal cell bodies on the dura were observed, consistent with the lack of neurons on this surface.", "No labeled neuronal cell bodies on the dura were observed, consistent with the lack of neurons on this surface. In contrast, we did find labeled cell bodies in the trigeminal ganglion ( Figure 4M ). No infection was seen in the CNS, even at 4 dpi, consistent with the lack of inputs from the brain into the trigeminal ganglion (N = 4 animals).", "No infection was seen in the CNS, even at 4 dpi, consistent with the lack of inputs from the brain into the trigeminal ganglion (N = 4 animals). To further characterize patterns and kinetics of viral transmission and directional specificity of transsynaptic spread, injections of rVSV(RABV-G) were made into the CP ( Figure 5A ). In order to determine which cells were labeled by direct uptake of virus in the inoculum, a separate set of animals were injected into the CP with the replication-incompetent rVSV G(RABV-G) (N = 3 animals, analyzed 3 dpi).", "In order to determine which cells were labeled by direct uptake of virus in the inoculum, a separate set of animals were injected into the CP with the replication-incompetent rVSV G(RABV-G) (N = 3 animals, analyzed 3 dpi). Cells labeled by rVSV G(RABV-G) were observed in the CP, GP, substantia nigra (SN), thalamus, and layers 3 and 5 of the cortex, consistent with infection at the axon terminal and retrograde labeling of cell bodies of neurons known to project directly to the CP ( Figure 5C ) (Albin et al., 1995) . Areas labeled by CP injection are indicated in Figure 5B .", "Areas labeled by CP injection are indicated in Figure 5B . The patterns of spread for the replication-competent rVSV(RABV-G) were characterized over the course of 1-5 dpi ( Figures 5D-H) . During this interval, progressively more cells in infected regions were labeled by rVSV(RABV-G), including within the CP, nucleus basalis, cortex, and GP (listed in Figure 5B ).", "During this interval, progressively more cells in infected regions were labeled by rVSV(RABV-G), including within the CP, nucleus basalis, cortex, and GP (listed in Figure 5B ). In addition, more cortical cells were labeled in clusters near cortical pyramidal neurons, both ipsilateral and contralateral to the injected side, including neurogliaform cells (data not shown). These data are in contrast to those observed following infection with an anterograde transsynaptic tracing virus, such as rVSV with its own G gene, rVSV(VSV-G) (Figure 5B) .", "These data are in contrast to those observed following infection with an anterograde transsynaptic tracing virus, such as rVSV with its own G gene, rVSV(VSV-G) (Figure 5B) . At 3 dpi following rVSV(VSV-G) injection into the CP, the cerebral cortex was not labeled, but regions receiving projections from the CP, such as the STn, GP, and SN, were labeled (Beier et al., 2011) . In order to investigate other areas for evidence of cell-to-cell retrograde transsynaptic spread, the nucleus basalis was examined following infection of the CP with replication-competent rVSV(RABV-G).", "In order to investigate other areas for evidence of cell-to-cell retrograde transsynaptic spread, the nucleus basalis was examined following infection of the CP with replication-competent rVSV(RABV-G). The nucleus basalis was labeled by 2 dpi (Figures 5E-H) , consistent with at least a single transsynaptic jump, as this area does not directly project to the CP. The virus appeared to travel transsynaptically at the rate of roughly 1 synapse per day, as evidenced by the lack of labeled neurogliaform cells in the cortex, and lack of neurons in the nucleus basalis at 1 dpi, and label appearing in these cell types/areas at 2 dpi, as previously observed (Beier et al., 2011) .", "The virus appeared to travel transsynaptically at the rate of roughly 1 synapse per day, as evidenced by the lack of labeled neurogliaform cells in the cortex, and lack of neurons in the nucleus basalis at 1 dpi, and label appearing in these cell types/areas at 2 dpi, as previously observed (Beier et al., 2011) . Labeling remained well-restricted to the expected corticostriatal circuits at 5 dpi, suggesting that viral spread becomes less efficient after crossing one or two connections, consistent with injections into V1 (Figure 4) . While glial cells can be infected and were observed near the injection site (van den Pol et al., 2002; Chauhan et al., 2010) , infected glial cells away from the injection site generally were not observed.", "While glial cells can be infected and were observed near the injection site (van den Pol et al., 2002; Chauhan et al., 2010) , infected glial cells away from the injection site generally were not observed. One advantage of having both anterograde and retrograde forms of the same virus is that they can be used in parallel, or in tandem, to trace circuitry to and from a single or multiple sites of injection, with each virus having similar kinetics of spread and gene expression. In fact, if different fluorophores are used in different viruses, e.g., rVSV(VSV-G) and rVSV(RABV-G), then the viruses can be co-injected into the same site and their transmission can be traced independently ( Figure 6A ).", "In fact, if different fluorophores are used in different viruses, e.g., rVSV(VSV-G) and rVSV(RABV-G), then the viruses can be co-injected into the same site and their transmission can be traced independently ( Figure 6A ). This is most straightforward if there are no cells at the injection site that are initially infected by both viruses. Co-infected cells can be easily detected, as they would express both fluorescent proteins shortly after injection.", "Co-infected cells can be easily detected, as they would express both fluorescent proteins shortly after injection. In order to determine whether two viruses would allow simultaneous anterograde and retrograde transsynaptic tracing from a single injection site, a rVSV(VSV-G) expressing Venus and a rVSV(RABV-G) expressing mCherry were injected individually (Figures 6B-D) or co-injected (Figures 6E-G) into the motor cortex, and brains were examined 3 dpi. The pattern of labeling from the co-injected brains was equivalent to the patterns observed when each virus was injected individually: rVSV(VSV-G) was observed to infect neurons in the cortex, CP, and downstream nuclei, whereas the rVSV(RABV-G) was not observed to infect neurons in the CP, but rather in the thalamus and nucleus basalis (N = 4).", "The pattern of labeling from the co-injected brains was equivalent to the patterns observed when each virus was injected individually: rVSV(VSV-G) was observed to infect neurons in the cortex, CP, and downstream nuclei, whereas the rVSV(RABV-G) was not observed to infect neurons in the CP, but rather in the thalamus and nucleus basalis (N = 4). The initial co-infection rate is dependent upon The presence or absence of labeling is indicated by (+) and (−), respectively. The extent of labeling is indicated by the number of (+).", "The extent of labeling is indicated by the number of (+). Some animals were infected with G viruses to determine which areas were labeled by direct uptake of the virions, rather than by replication and transmission. These were sacrificed at 3 dpi.", "These were sacrificed at 3 dpi. (C) Parasaggital section of a brain infected with VSV[greek delta]G(RABV-G). The injection site is marked by a red arrow.", "The injection site is marked by a red arrow. Several areas that project directly to the CP were labeled due to direct uptake of the virions, including the cortex, thalamus, and GP (arrowheads), 3 dpi. the dose of the initial inocula.", "the dose of the initial inocula. When injecting 3 × 10 3 focus forming units (ffu) rVSV(VSV-G) and 3 × 10 4 ffu rVSV(RABV-G), no co-infection was observed at the injection site. Thus, co-infection of the same brain region, without co-infection of the same cells,\n\ndoes not alter the spreading behavior of either rVSV(VSV-G) or rVSV(RABV-G).", "Thus, co-infection of the same brain region, without co-infection of the same cells,\n\ndoes not alter the spreading behavior of either rVSV(VSV-G) or rVSV(RABV-G). One example of how this dual retrograde and anterograde transsynaptic tracing system can be used is to determine if three Frontiers in Neural Circuits February 2013 | Volume 7 | Article 11 | 8 distinct regions are connected and the directionality of any connections. For example, the anterograde transsynaptic virus can be injected into one region, the retrograde into another, and a third region can then be examined for evidence of labeling by either or both viruses (e.g., Figure 6H ).", "For example, the anterograde transsynaptic virus can be injected into one region, the retrograde into another, and a third region can then be examined for evidence of labeling by either or both viruses (e.g., Figure 6H ). To test this possibility, rVSV(VSV-G) was injected into the motor cortex, rVSV(RABV-G) was injected into the substantia nigra pars reticulara (SNr), and animals were sacrificed at 3 dpi. We observed that cells were singly labeled, either with Venus [rVSV(VSV-G)] or with mCherry [rVSV(RABV-G)], and were located largely in different regions of the CP (Figures 6I,J) (N = 3) .", "We observed that cells were singly labeled, either with Venus [rVSV(VSV-G)] or with mCherry [rVSV(RABV-G)], and were located largely in different regions of the CP (Figures 6I,J) (N = 3) . These results suggest that the anterograde connections from the cells infected with rVSV(VSV-G) in the M1 were with CP MSNs that did not project to the region of the SNr injected with rVSV(RABV-G) (N = 3 animals). In addition to polysynaptic tracing, VSV can be modified to trace circuits monosynaptically (Beier et al., 2011) .", "In addition to polysynaptic tracing, VSV can be modified to trace circuits monosynaptically (Beier et al., 2011) . With RABV, this was achieved in vivo by first infecting with an adeno-associated virus (AAV) expressing TVA, a receptor for an avian retrovirus, and RABV-G (Wall et al., 2010) . This was followed 3 weeks later by infection with a G RABV with an EnvA/RABV-G chimeric glycoprotein on the virion surface (Wickersham et al., 2007b) , which allowed infection specifically of the cells expressing TVA.", "This was followed 3 weeks later by infection with a G RABV with an EnvA/RABV-G chimeric glycoprotein on the virion surface (Wickersham et al., 2007b) , which allowed infection specifically of the cells expressing TVA. A similar strategy was used to test rVSV's ability to monosynaptically trace retrogradely connected neurons in vivo. Inputs to choline acetyltransferase (ChAT)-expressing neurons in the striatum were used for this test.", "Inputs to choline acetyltransferase (ChAT)-expressing neurons in the striatum were used for this test. These neurons primarily receive input from the cortex and the thalamus (Thomas et al., 2000; Bloomfield et al., 2007) (Figure 7A) . In order to mark this population, we crossed ChAT-Cre mice to Ai9 mice, which express tdTomato in cells with a Cre expression history (Madisen et al., 2010) .", "In order to mark this population, we crossed ChAT-Cre mice to Ai9 mice, which express tdTomato in cells with a Cre expression history (Madisen et al., 2010) . Six-week-old mice from this cross were injected in the CP with two AAV vectors: one expressing a Cre-conditional (\"floxed\") TVA-mCherry fusion protein, and another expressing a floxed RABV-G. Two weeks later, the mice were injected in the same coordinates with rVSV G with the EnvA/RABV-G chimeric glycoprotein on the virion surface [rVSV G(A/RG)] (Beier et al., 2011) . Cells successfully infected with these two AAV vectors could host infection by a rVSV and should be able to produce rVSV virions with RABV-G on the surface.", "Cells successfully infected with these two AAV vectors could host infection by a rVSV and should be able to produce rVSV virions with RABV-G on the surface. Such starter cells should also express tdTomato and GFP. If rVSV were to be produced, and if it were to transmit across the synapse retrogradely, cortical and thalamic neurons should be labeled by GFP.", "If rVSV were to be produced, and if it were to transmit across the synapse retrogradely, cortical and thalamic neurons should be labeled by GFP. Mice injected with these AAV and rVSV viruses were sacrificed 5 days after rVSV infection, and brains analyzed for fluorescence. As expected for starter cells, some neurons in the CP expressed both tdTomato and GFP (Figure 7B) .", "As expected for starter cells, some neurons in the CP expressed both tdTomato and GFP (Figure 7B) . Outside of the CP, small numbers of GFP+ neurons that were not mCherry+ were observed in the cortex (Figures 7C,D) and thalamus (Figure 7E) , consistent with retrograde spread. Control animals not expressing Cre, or not injected with AAV encoding RABV-G, did not label cells in the cortex or thalamus (N = 3 for both controls and experimental condition).", "Control animals not expressing Cre, or not injected with AAV encoding RABV-G, did not label cells in the cortex or thalamus (N = 3 for both controls and experimental condition). Here, we report on the use of rVSV as a retrograde transsynaptic tracer for CNS circuitry. VSV can be modified to encode the RABV-G protein in the viral genome, allowing the virus to replicate and transmit across multiple synaptically connected cells, i.e., as a polysynaptic tracer.", "VSV can be modified to encode the RABV-G protein in the viral genome, allowing the virus to replicate and transmit across multiple synaptically connected cells, i.e., as a polysynaptic tracer. Alternatively, if the virus has the G gene deleted from its genome and RABV-G is provided in trans, it behaves as a monosynaptic tracer (Beier et al., 2011) . Although it has been known for many years that RABV travels retrogradely among neurons (Astic et al., 1993; Ugolini, 1995; Kelly and Strick, 2003) , and pseudotyping lentiviruses with RABV-G is sufficient for axonal transport (Mazarakis et al., 2001) , the retrograde transmission specificity among neurons had not been clearly shown to be a property of the G protein itself, as it might have been due to other viral proteins in addition to, or instead of, the viral G protein.", "Although it has been known for many years that RABV travels retrogradely among neurons (Astic et al., 1993; Ugolini, 1995; Kelly and Strick, 2003) , and pseudotyping lentiviruses with RABV-G is sufficient for axonal transport (Mazarakis et al., 2001) , the retrograde transmission specificity among neurons had not been clearly shown to be a property of the G protein itself, as it might have been due to other viral proteins in addition to, or instead of, the viral G protein. Since native VSV does not have these retrograde transsynaptic properties (van den Pol et al., 2002; Beier et al., 2011) , and the only alteration to the VSV genome was the substitution of the VSV G gene with the G gene of RABV, it is clear that the RABV glycoprotein is responsible for retrograde direction of viral transmission across synapses, at least in the case of rVSV. The early onset of gene expression from VSV relative to RABV (one hour vs. multiple hours) makes it beneficial in experimental paradigms in which the experiment needs to be done within a narrow window of time, such as tissue slices and explants.", "The early onset of gene expression from VSV relative to RABV (one hour vs. multiple hours) makes it beneficial in experimental paradigms in which the experiment needs to be done within a narrow window of time, such as tissue slices and explants. In addition, more than one transgene can be encoded in the viral genome without the need of a 2A or IRES element. The use of the first position of the genome enhances the expression level of the transgene inserted at that location, since VSV (and RABV) express genes in a transcriptional gradient; therefore, the first gene is the most highly transcribed (Knipe, 2007) .", "The use of the first position of the genome enhances the expression level of the transgene inserted at that location, since VSV (and RABV) express genes in a transcriptional gradient; therefore, the first gene is the most highly transcribed (Knipe, 2007) . This leads to rational predictions of expression levels so that one can choose the position of insertion of a transgene, or transgenes, according to this gradient and the desired level of expression. The size of the viral capsid is apparently not rigid, allowing for the inclusion of genomes that are substantially larger than the native genome, unlike the rigid capacity for some other viral vectors, such as AAV Yan et al., 2000) .", "The size of the viral capsid is apparently not rigid, allowing for the inclusion of genomes that are substantially larger than the native genome, unlike the rigid capacity for some other viral vectors, such as AAV Yan et al., 2000) . The fact that VSV can be made to spread anterogradely (Beier et al., 2011) or retrogradely across synapses with the change of a single gene affords several advantages over viral tracers that heretofore have not shown such flexibility in the directionality of tracing. In addition to the obvious application of tracing anterograde connections, combinations can be made to exploit the different forms of the virus.", "In addition to the obvious application of tracing anterograde connections, combinations can be made to exploit the different forms of the virus. One example that employs the simultaneous infection with an anterograde and retrograde form of VSV is demonstrated in Figure 6 . This experiment was designed to address whether the anterograde projections from the cortex to the CP would label the same brain regions as were labeled by a retrograde virus injected into the SN.", "This experiment was designed to address whether the anterograde projections from the cortex to the CP would label the same brain regions as were labeled by a retrograde virus injected into the SN. Although a block of superinfection by the virus may preclude infection of the same cell with multiple rVSVs, adjacent cells could still become labeled by different viruses (Whitaker-Dowling et al., 1983) . The observed results could be due to a preferential labeling by the anterograde transsynaptic virus of indirect pathway MSNs in this experiment, which then synapse onto the GP, thereby reflecting a viral bias.", "The observed results could be due to a preferential labeling by the anterograde transsynaptic virus of indirect pathway MSNs in this experiment, which then synapse onto the GP, thereby reflecting a viral bias. Alternatively, it could indicate that the cortical neurons in the injected region largely do not label the MSNs that project to the area of the SN injected with the retrograde virus. One further possibility is that too little virus was used to observe co-labeling of a given region.", "One further possibility is that too little virus was used to observe co-labeling of a given region. However, given the density of infection (i.e., Figures 6I,J) , the latter possibility seems unlikely. Additionally, the spread of the polysynaptic rVSV(RABV-G) appears to attenuate with increasing numbers of synapses crossed, permitting an analysis of more restricted viral spread.", "Additionally, the spread of the polysynaptic rVSV(RABV-G) appears to attenuate with increasing numbers of synapses crossed, permitting an analysis of more restricted viral spread. This is quite fortuitous, as if spread were to continue, it would lead to widespread infection and lethality. In addition, reconstruction of connectivity would be more difficult.", "In addition, reconstruction of connectivity would be more difficult. This reduced efficiency appears to also hold for the monosynaptic form of VSV complemented with RABV-G, as the efficiency of transmission appeared lower than the comparable experiment with RABV (Watabe-Uchida et al., 2012) . This is likely due to viral attenuation when VSV-G is replaced with RABV-G.\n\nWe were attracted to the use of VSV as a viral tracer due to its long track record as a safe, replication-competent laboratory agent.", "This is likely due to viral attenuation when VSV-G is replaced with RABV-G.\n\nWe were attracted to the use of VSV as a viral tracer due to its long track record as a safe, replication-competent laboratory agent. Laboratory workers using VSV have not contracted any diseases, and natural VSV infections among human populations in Central America and the southwestern United States (Rodríguez, 2002) occur without evident pathology (Johnson et al., 1966; Brody et al., 1967) . VSV was thus an attractive candidate for its use as a polysynaptic tracer for CNS studies, which requires an ability to replicate through multiple transmission cycles.", "VSV was thus an attractive candidate for its use as a polysynaptic tracer for CNS studies, which requires an ability to replicate through multiple transmission cycles. Both replicationcompetent and incompetent forms of VSV are in use under Biosafety Level 2 containment. Replication-competent RABV is Biosafety Level 3, due to the fact that infection with replicationcompetent RABV is almost always fatal to humans and in mice when infected intracerebrally (Smith, 1981; Knipe, 2007) .", "Replication-competent RABV is Biosafety Level 3, due to the fact that infection with replicationcompetent RABV is almost always fatal to humans and in mice when infected intracerebrally (Smith, 1981; Knipe, 2007) . Differences in pathogenicity between VSV and RABV are likely due to the ability of RABV to evade the innate immune system, particularly interferon (Hangartner et al., 2006; Junt et al., 2007; Lyles and Rupprecht, 2007; Rieder and Conzelmann, 2009; Iannacone et al., 2010) . VSV infection efficiently triggers an interferon response, and it has not evolved a method of escape from this response, unlike RABV (Brzózka et al., 2006) .", "VSV infection efficiently triggers an interferon response, and it has not evolved a method of escape from this response, unlike RABV (Brzózka et al., 2006) . In fact, VSV is being pursued as a vaccine for other viruses, including RABV (Lichty et al., 2004; Publicover et al., 2004; Kapadia et al., 2005; Schwartz et al., 2007; Iyer et al., 2009; Geisbert and Feldmann, 2011) . VSV does not typically spread beyond the initially infected site in the periphery (Kramer et al., 1983; Vogel and Fertsch, 1987) .", "VSV does not typically spread beyond the initially infected site in the periphery (Kramer et al., 1983; Vogel and Fertsch, 1987) . This likely is the cause of the minor or absent symptoms in humans and animals infected in nature. Polysynaptic VSV vectors are thus predicted to be much safer than polysynaptic RABV vectors.", "Polysynaptic VSV vectors are thus predicted to be much safer than polysynaptic RABV vectors. We have tested this prediction by injecting a series of mice in the footpads and hind leg muscles with rVSV(RABV-G), with the result that no injected animals showed any evidence of morbidity or mortality (Beier, Goz et al., in preparation) . While safer for laboratory workers than RABV, the main drawback to using VSV is its rapid cellular toxicity (van den Pol et al., 2009; Beier et al., 2011) .", "While safer for laboratory workers than RABV, the main drawback to using VSV is its rapid cellular toxicity (van den Pol et al., 2009; Beier et al., 2011) . Toxicity is due to suppression of cellular transcription and a block in the export of cellular RNAs from the nucleus to the cytoplasm (Black and Lyles, 1992; Her et al., 1997; Ahmed and Lyles, 1998; Petersen et al., 2000; von Kobbe et al., 2000) , as well as inhibition of the translation of cellular mRNAs (Francoeur et al., 1987; Jayakar et al., 2000; Kopecky et al., Frontiers in Neural Circuits February 2013 | Volume 7 | Article 11 | 10 2001). VSV is much quicker to enact its gene expression program than is RABV, such that cells suffer the toxic effects more quickly than after RABV infection.", "VSV is much quicker to enact its gene expression program than is RABV, such that cells suffer the toxic effects more quickly than after RABV infection. One aspect of VSV that can be exploited in the future to ameliorate the speed of toxicity is the use of VSV mutants and variants. One such mutant is the M51R, which permitted us to conduct physiological analyses of pre-and post-synaptic cells (Beier et al., 2011) .", "One such mutant is the M51R, which permitted us to conduct physiological analyses of pre-and post-synaptic cells (Beier et al., 2011) . We are in the process of examining the transmission properties of this mutant in vivo, as well as the effects of other mutations or viral variants on prolonging the health of neurons after infection. rVSV vectors can be used to study the connectivity of neuronal circuitry.", "rVSV vectors can be used to study the connectivity of neuronal circuitry. In addition to combinations of replication-competent forms of VSV, the replication-incompetent, monosynaptic forms of the virus can be easily combined, without the need to change viruses (Beier et al., 2011) . This allows a straightforward way to study both the projections into, and out from, a genetically defined cell population.", "This allows a straightforward way to study both the projections into, and out from, a genetically defined cell population. This can be done with the same viral genome, with the only change needed being the glycoprotein, for the selection of the direction of transmission. This flexibility of VSV makes it a powerful, multi-application vector for studying connectivity in the CNS.", "This flexibility of VSV makes it a powerful, multi-application vector for studying connectivity in the CNS. All rVSV clones were cloned from the rVSV G backbone (Chandran et al., 2005) . mCherry, Kusabira orange, Venus, and CFP were cloned into the first (GFP) position using XhoI and MscI sites, and VSV-G (a gift from Richard Mulligan, Harvard Medical School, Boston, MA) and RABV-G (a gift from Ed Callaway, Salk Institute, San Diego, CA) were cloned into the fifth (G) position using the MluI and NotI restriction sites.", "mCherry, Kusabira orange, Venus, and CFP were cloned into the first (GFP) position using XhoI and MscI sites, and VSV-G (a gift from Richard Mulligan, Harvard Medical School, Boston, MA) and RABV-G (a gift from Ed Callaway, Salk Institute, San Diego, CA) were cloned into the fifth (G) position using the MluI and NotI restriction sites. Genes for fluorescent proteins were obtained from Clontech. Viruses were rescued as previously described (Whelan et al., 1995) .", "Viruses were rescued as previously described (Whelan et al., 1995) . At 95% confluency, eight 10 cm plates of BSR cells were infected at an MOI of 0.01. Viral supernatants were collected at 24-h time intervals and ultracentrifuged at 21,000 RPM using a SW28 rotor and resuspended in 0.2% of the original volume.", "Viral supernatants were collected at 24-h time intervals and ultracentrifuged at 21,000 RPM using a SW28 rotor and resuspended in 0.2% of the original volume. For titering, concentrated viral stocks were applied in a dilution series to 100% confluent BSR cells and plates were examined at 12 hpi. Viral stocks were stored at −80 • C.\n\nFor G viruses, 293T cells were transfected with PEI (Ehrhardt et al., 2006) at 70% confluency on 10 cm dishes with 5 µg of pCAG-RABV-G. Twenty-four hours post-infection, the cells were infected at an MOI of 0.01 with rVSV G expressing either GFP or mCherry.", "Viral stocks were stored at −80 • C.\n\nFor G viruses, 293T cells were transfected with PEI (Ehrhardt et al., 2006) at 70% confluency on 10 cm dishes with 5 µg of pCAG-RABV-G. Twenty-four hours post-infection, the cells were infected at an MOI of 0.01 with rVSV G expressing either GFP or mCherry. Viral supernatants were collected for the subsequent 4 days at 24 h intervals. Virus preparations are now available from the Salk GT3 viral core ( All plasmids are available from Addgene (\n\nAAV-FLEx-RABV-G and AAV-FLEx-TVA-mCherry plasmids originated from the Lab of Naoshige Uchida (Watabe-Uchida et al., 2012) , and virus stocks were generous gifts from Brad Lowell, Harvard Medical School.", "Virus preparations are now available from the Salk GT3 viral core ( All plasmids are available from Addgene (\n\nAAV-FLEx-RABV-G and AAV-FLEx-TVA-mCherry plasmids originated from the Lab of Naoshige Uchida (Watabe-Uchida et al., 2012) , and virus stocks were generous gifts from Brad Lowell, Harvard Medical School. ChAT-Cre (B6;129S6-Chat tm1(cre)Lowl /J) and Ai9 (B6.Cg-Gt(ROSA)26Sor<tm9(CAG-tdTomato)Hze>/J) mice were obtained from the Jackson Laboratory (Madisen et al., 2010) . Eight-week-old CD-1 mice were injected using pulled capillary microdispensers (Drummond Scientific, Cat.", "Eight-week-old CD-1 mice were injected using pulled capillary microdispensers (Drummond Scientific, Cat. No: 5-000-2005) , using coordinates from The Mouse Brain in Stereotaxic Coordinates (Franklin and Paxinos, 1997) . Injection coordinates (in mm) used were: For multi-color analysis (Figures 1C,D) , 3 × 10 9 ffu/mL rVSV was injected into various regions.", "Injection coordinates (in mm) used were: For multi-color analysis (Figures 1C,D) , 3 × 10 9 ffu/mL rVSV was injected into various regions. For CP injections, 100 nL of rVSV(RABV-G) or rVSV(VSV-G) at 3 × 10 7 ffu/mL was injected at a rate of 100 nL/min. For the replication-incompetent viruses, 100 nL of 1 × 10 7 ffu/mL rVSV G(RABV-G) or rVSV G (VSV-G) was injected.", "For the replication-incompetent viruses, 100 nL of 1 × 10 7 ffu/mL rVSV G(RABV-G) or rVSV G (VSV-G) was injected. In the motor cortex, 100 nL of 1 × 10 7 ffu/mL rVSV(RABV-G) was injected, and mice harvested 2 dpi. For V1 injections, 100 nL of 3 × 10 10 ffu/mL rVSV(RABV-G) was injected, and mice were examined 3 or 7 dpi.", "For V1 injections, 100 nL of 3 × 10 10 ffu/mL rVSV(RABV-G) was injected, and mice were examined 3 or 7 dpi. For infections of the dura mater, 1 µL of 3 × 10 10 ffu/mL rVSV(RABV-G) was applied to the surface of the dura. The virus was allowed to absorb, and the surface was subsequently covered in bone wax, and the wound sutured.", "The virus was allowed to absorb, and the surface was subsequently covered in bone wax, and the wound sutured. For co-injections of virus into the same animal, 100 nL of a combination of 3 × 10 7 ffu/mL rVSV(VSV-G) and 3 × 10 8 ffu/mL rVSV(RABV-G) were co-injected into the motor cortex, and brains examined 3 dpi. For injections of the viruses into different regions, 100 nL of 3 × 10 7 ffu/mL rVSV(VSV-G) was injected into M1, and 100 nL of 3 × 10 8 ffu/mL rVSV(RABV-G) into the SNr, and brains examined 3 dpi.", "For injections of the viruses into different regions, 100 nL of 3 × 10 7 ffu/mL rVSV(VSV-G) was injected into M1, and 100 nL of 3 × 10 8 ffu/mL rVSV(RABV-G) into the SNr, and brains examined 3 dpi. A lower titer of rVSV(VSV-G) was used, as rVSV(RABV-G) is attenuated. All mouse work was conducted in biosafety containment level 2 conditions and was approved by the Longwood Medical Area Institutional Animal Care and Use Committee.", "All mouse work was conducted in biosafety containment level 2 conditions and was approved by the Longwood Medical Area Institutional Animal Care and Use Committee. Recordings were made from cortical pyramidal neurons in slices taken from postnatal day 12-18 mice, inoculated in the CP 12-18 h prior with rVSV(RABV-G). Coronal slices (300 µm thick) were cut in ice-cold external solution containing (in mM): 110 choline, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 2.5 KCl, 7 MgCl 2 , 0.5 CaCl 2 , 25 glucose, 11.6 Na-ascorbate, and 3.1 Na-pyruvate, bubbled with 95% O 2 and 5% CO 2 .", "Coronal slices (300 µm thick) were cut in ice-cold external solution containing (in mM): 110 choline, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 2.5 KCl, 7 MgCl 2 , 0.5 CaCl 2 , 25 glucose, 11.6 Na-ascorbate, and 3.1 Na-pyruvate, bubbled with 95% O 2 and 5% CO 2 . Slices were then transferred to artificial cerebrospinal fluid (ACSF) containing (in mM): 127 NaCl, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 2.5 KCl, 1 MgCl 2 , 2 CaCl 2 , and 25 glucose, bubbled with 95% O 2 and 5% CO 2 . After an incubation period Frontiers in Neural Circuits February 2013 | Volume 7 | Article 11 | 11 of 30-40 min at 34 • C, slices were stored at room temperature.", "After an incubation period Frontiers in Neural Circuits February 2013 | Volume 7 | Article 11 | 11 of 30-40 min at 34 • C, slices were stored at room temperature. All experiments were conducted at room temperature (25 • C). In all experiments, 50 µM picrotoxin, 10 µM 2,3-Dioxo-6-nitro-1, 2, 3, 4 -tetrahydrobenzo [f]quinoxaline -7-sulfonamide (NBQX), and 10 µM 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) were present in the ACSF to block GABAA/C, AMPA, and NMDA receptor-mediated transmission, respectively.", "In all experiments, 50 µM picrotoxin, 10 µM 2,3-Dioxo-6-nitro-1, 2, 3, 4 -tetrahydrobenzo [f]quinoxaline -7-sulfonamide (NBQX), and 10 µM 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) were present in the ACSF to block GABAA/C, AMPA, and NMDA receptor-mediated transmission, respectively. All chemicals were from Sigma or Tocris. Whole-cell recordings were obtained from infected and uninfected deep layer cortical pyramidal neurons identified with video-IR/DIC and GFP fluorescence was detected using epifluorescence illumination.", "Whole-cell recordings were obtained from infected and uninfected deep layer cortical pyramidal neurons identified with video-IR/DIC and GFP fluorescence was detected using epifluorescence illumination. With the deep layers of the cortex, 2-photon laser scanning microscopy (2PLSM) was used to confirm the cell types based on morphology. Deep layer pyramidal neurons had large cell bodies, classic pyramidal shape and dendritic spines.", "Deep layer pyramidal neurons had large cell bodies, classic pyramidal shape and dendritic spines. Glass electrodes (2-4 M ) were filled with internal solution containing (in mM): 135 KMeSO 4 , 5 KCl, 5 HEPES, 4 MgATP, 0.3 NaGTP, 10 Na 2 HPO 4 , 1 EGTA, and 0.01 Alexa Fluor-594 (to image neuronal morphology) adjusted to pH 7.4 with KOH. Current and voltage recordings were made at room temperature using a AxoPatch 200B or a Multiclamp 700B amplifier.", "Current and voltage recordings were made at room temperature using a AxoPatch 200B or a Multiclamp 700B amplifier. Data was filtered at 5 kHz and digitized at 10 kHz. Imaging and physiology data were acquired and analyzed as described previously (Carter and Sabatini, 2004) .", "Imaging and physiology data were acquired and analyzed as described previously (Carter and Sabatini, 2004) . Resting membrane potential was determined by the average of three 5-s sweeps with no injected current. Passive properties of the cell, membrane (Rm) and series resistance (Rs) and capacitance (Cm), were measured while clamping cells at −65 mV and applying voltage steps from −55 to −75 mV.", "Passive properties of the cell, membrane (Rm) and series resistance (Rs) and capacitance (Cm), were measured while clamping cells at −65 mV and applying voltage steps from −55 to −75 mV. The current-firing relationship was determined in current clamp with 1-s periods of injected current from 100 to 500 pA. The time course of viral gene expression experiments were carried out in organotypic hippocampal slice cultures prepared from postnatal day 5-7 Sprague-Dawley rats as described previously (Stoppini et al., 1991) .", "The time course of viral gene expression experiments were carried out in organotypic hippocampal slice cultures prepared from postnatal day 5-7 Sprague-Dawley rats as described previously (Stoppini et al., 1991) . Slices were infected after 7 days in vitro, and images were acquired on a two-photon microscope." ]

[ "Defining the connections among neurons is critical to our understanding of the structure and function of the nervous system. Recombinant viruses engineered to transmit across synapses provide a powerful approach for the dissection of neuronal circuitry in vivo. We recently demonstrated that recombinant vesicular stomatitis virus (VSV) can be endowed with anterograde or retrograde transsynaptic tracing ability by providing the virus with different glycoproteins.", "We recently demonstrated that recombinant vesicular stomatitis virus (VSV) can be endowed with anterograde or retrograde transsynaptic tracing ability by providing the virus with different glycoproteins. Here we extend the characterization of the transmission and gene expression of recombinant VSV (rVSV) with the rabies virus glycoprotein (RABV-G), and provide examples of its activity relative to the anterograde transsynaptic tracer form of rVSV. rVSV with RABV-G was found to drive strong expression of transgenes and to spread rapidly from neuron to neuron in only a retrograde manner.", "rVSV with RABV-G was found to drive strong expression of transgenes and to spread rapidly from neuron to neuron in only a retrograde manner. Depending upon how the RABV-G was delivered, VSV served as a polysynaptic or monosynaptic tracer, or was able to define projections through axonal uptake and retrograde transport. In animals co-infected with rVSV in its anterograde form, rVSV with RABV-G could be used to begin to characterize the similarities and differences in connections to different areas.", "In animals co-infected with rVSV in its anterograde form, rVSV with RABV-G could be used to begin to characterize the similarities and differences in connections to different areas. rVSV with RABV-G provides a flexible, rapid, and versatile tracing tool that complements the previously described VSV-based anterograde transsynaptic tracer. Text: Mapping neuronal connectivity in the central nervous system (CNS) of even simple organisms is a difficult task.", "Text: Mapping neuronal connectivity in the central nervous system (CNS) of even simple organisms is a difficult task. Recombinant viruses engineered to trace synaptic connections and express transgenes promise to enable higher-throughput mapping of connections among neurons than other methods, e.g., serial reconstruction from electron micrographs (Bock et al., 2011; Briggman et al., 2011) . The Pseudorabies (PRV) and Rabies viruses (RABV) have been the best characterized and most utilized circuit tracing viruses to date (Ugolini et al., 1989; Kelly and Strick, 2000) .", "The Pseudorabies (PRV) and Rabies viruses (RABV) have been the best characterized and most utilized circuit tracing viruses to date (Ugolini et al., 1989; Kelly and Strick, 2000) . RABV was recently modified by Wickersham and colleagues such that it can travel across only one synapse, allowing for a straightforward definition of monosynaptic connections (Wickersham et al., 2007b) . This strategy permitted the first unambiguous identification of retrogradely connected cells from an initially infected cell (\"starter cell\"), without the need for electrophysiology.", "This strategy permitted the first unambiguous identification of retrogradely connected cells from an initially infected cell (\"starter cell\"), without the need for electrophysiology. Moreover, the starter cell could be defined through the expression of a specific viral receptor that limited the initial infection. Recently, we created an anterograde monosynaptic virus that complements the previously available retrograde viral tracers (Beier et al., 2011) .", "Recently, we created an anterograde monosynaptic virus that complements the previously available retrograde viral tracers (Beier et al., 2011) . Vesicular stomatitis virus (VSV), a virus related to RABV, with its own glycoprotein (G) gene (VSV-G), or with a G from the unrelated lymphocytic choriomeningitis virus (LCMV-G), spreads in the anterograde direction across synapses. VSV can be used as a polysynaptic tracer that spreads across many synapses, owing to the fact that the normal, replicationcompetent form of the virus does not cause serious diseases in humans (Brandly and Hanson, 1957; Johnson et al., 1966; Brody et al., 1967) .", "VSV can be used as a polysynaptic tracer that spreads across many synapses, owing to the fact that the normal, replicationcompetent form of the virus does not cause serious diseases in humans (Brandly and Hanson, 1957; Johnson et al., 1966; Brody et al., 1967) . Whether the virus is a monosynaptic or polysynaptic tracer is determined by the method of delivery of the G gene ( Figure 1A) . Advantages of VSV are that it is well-characterized, is relatively simple in comparison to PRV, and it rapidly grows to high titer in tissue culture cells.", "Advantages of VSV are that it is well-characterized, is relatively simple in comparison to PRV, and it rapidly grows to high titer in tissue culture cells. It is also being developed as a vaccine vector, often using a G of another virus as the immunogen, as well as being developed as a cytocidal agent that will target tumor cells in humans (Balachandran and Barber, 2000; Stojdl et al., 2000 Stojdl et al., , 2003 . Previous studies of the anatomical patterns of transmission, as well as physiological recordings, have shown that the transmission of VSV and RABV among neurons is via synapses (Kelly and Strick, 2000; Wickersham et al., 2007b; Beier et al., 2011) .", "Previous studies of the anatomical patterns of transmission, as well as physiological recordings, have shown that the transmission of VSV and RABV among neurons is via synapses (Kelly and Strick, 2000; Wickersham et al., 2007b; Beier et al., 2011) . In addition, it has been shown that RABV, as well as lentiviruses with RABV-G in their envelope, travel retrogradely from an injection site (Mazarakis et al., 2001; Wickersham et al., 2007a) . We hypothesized that providing a recombinant VSV (rVSV) with the RABV-G would create a retrograde polysynaptic transsynaptic tracer without the biosafety concerns inherent to RABV.", "We hypothesized that providing a recombinant VSV (rVSV) with the RABV-G would create a retrograde polysynaptic transsynaptic tracer without the biosafety concerns inherent to RABV. Our initial characterization of rVSV with RABV-G showed that indeed FIGURE 1 | Synaptic tracing strategies using VSV. (A) Schematic illustrating the strategies for polysynaptic or monosynaptic retrograde or anterograde transsynaptic transmission of rVSV encoding GFP.", "(A) Schematic illustrating the strategies for polysynaptic or monosynaptic retrograde or anterograde transsynaptic transmission of rVSV encoding GFP. The initially infected cell is indicated by an asterisk. VSV encoding a glycoprotein (G) within its genome can spread polysynaptically. The direction of the spread depends on the identity of the glycoprotein.", "The direction of the spread depends on the identity of the glycoprotein. Infected neurons are shown in green. In some cases, the initially infected starter cell can be defined by the expression of an avian receptor, TVA (tagged with a red fluorescent protein).", "In some cases, the initially infected starter cell can be defined by the expression of an avian receptor, TVA (tagged with a red fluorescent protein). The TVA-expressing neurons can then be specifically infected by rVSV G with the EnvA/RABV-G (A/RG) glycoprotein (Wickersham et al., 2007b) on the virion surface [rVSV G(A/RG)]. These starter cells are then yellow, due to viral GFP and mCherry from TVA-mCherry expression.", "These starter cells are then yellow, due to viral GFP and mCherry from TVA-mCherry expression. For monosynaptic tracing, the G protein is expressed in trans in the TVA-expressing cell, and thus complements rVSV G to allow transmission in a specific direction. (B) Genomic diagrams of rVSV vectors.", "(B) Genomic diagrams of rVSV vectors. All VSVs contain four essential proteins: N, P, M, and L. Some viruses encode a G gene in their genome, which allows them to spread polysynaptically. rVSV vectors typically encode a transgene in the first position, while others carry an additional transgene in the G position.", "rVSV vectors typically encode a transgene in the first position, while others carry an additional transgene in the G position. (C) Morphological characterization of rVSV-infected neurons in several locations within the mouse brain. (i,ii) Caudate-putamen (CP) neurons at 4 dpi from an injection of the CP with rVSV(VSV-G) viruses encoding (i) CFP or (ii) Korange.", "(i,ii) Caudate-putamen (CP) neurons at 4 dpi from an injection of the CP with rVSV(VSV-G) viruses encoding (i) CFP or (ii) Korange. (iii) Labeled neurons of the CA1 region of the hippocampus are shown at 5 dpi following injection into the hippocampus of rVSV(VSV-G) encoding Venus. (iv,v) Cortical pyramidal neurons are shown following injection into the CP of rVSV(RABV-G) expressing (iv) GFP at 24 hpi, or (v) mCherry at 48 hpi.", "(iv,v) Cortical pyramidal neurons are shown following injection into the CP of rVSV(RABV-G) expressing (iv) GFP at 24 hpi, or (v) mCherry at 48 hpi. Inset in (iv) is a high magnification of the neuron in panel (iv), highlighting labeling of dendritic spines. (vi) Multiple viruses can be co-injected into the same animal.", "(vi) Multiple viruses can be co-injected into the same animal. Here, individual rVSV G(VSV-G) viruses encoding CFP, GFP , Venus, Korange, and mCherry were used to infect the cortex. Scale bars = 50 µm.", "Scale bars = 50 µm. February 2013 | Volume 7 | Article 11 | 2 it could be taken up as a retrograde tracer (Beier et al., 2011) . To determine if it could transmit among neurons following its replication in neurons, and to further analyze the transmission patterns of both the monosynaptic and polysynaptic forms of rVSV with RABV-G, we made injections into several CNS and peripheral locations.", "To determine if it could transmit among neurons following its replication in neurons, and to further analyze the transmission patterns of both the monosynaptic and polysynaptic forms of rVSV with RABV-G, we made injections into several CNS and peripheral locations. In addition, we performed co-infections of rVSV with RABV-G and the anterograde form of rVSV in order to exploit the differences in the directionality of transmission of these two viruses in mapping circuits. Schematics of viruses created and used throughout this study are shown in Figure 1 .", "Schematics of viruses created and used throughout this study are shown in Figure 1 . We created rVSV vector plasmids carrying different transgenes in either the first or fifth genomic positions ( Figure 1B) . After rescuing each virus, we tested the ability of each to express transgenes in different brain regions through intracranial injections ( Figure 1C ).", "After rescuing each virus, we tested the ability of each to express transgenes in different brain regions through intracranial injections ( Figure 1C ). All rVSV vectors drove robust fluorophore expression 1 or 2 days post-infection (hpi) ( Figure 1C ) (van den Pol et al., 2009) . In fact, by 12 hpi, labeling was sufficiently bright to image fine morphological details, such as dendritic spines ( Figure 1C ,iv).", "In fact, by 12 hpi, labeling was sufficiently bright to image fine morphological details, such as dendritic spines ( Figure 1C ,iv). To characterize the physiological properties of cells infected with rVSV, we tested a replication-competent rVSV encoding GFP, with RABV-G in the genome in place of VSV-G [hereafter designated rVSV(RABV-G)]. van den Pol et al.", "van den Pol et al. reported that hippocampal neurons infected with replication-incompetent (G-deleted or \" G\") rVSV were physiologically healthy at 12-14 hpi, but were less so by 1 day post-infection (dpi) (van den Pol et al., 2009) . Given the known toxicity of both VSV and RABV-G (Coulon et al., 1982) , we tested the physiology of cortical pyramidal neurons in the motor cortex (M1) infected with rVSV(RABV-G).", "Given the known toxicity of both VSV and RABV-G (Coulon et al., 1982) , we tested the physiology of cortical pyramidal neurons in the motor cortex (M1) infected with rVSV(RABV-G). Between 12 and 18 hpi, the membrane capacitance, input resistance, resting membrane potential, and current-to-action potential firing relationship were indistinguishable between infected and uninfected neurons (Figure 2) . However, by 2 dpi, electrophysiological properties were so abnormal in the infected cortical pyramidal cells that physiological measurements could not be made.", "However, by 2 dpi, electrophysiological properties were so abnormal in the infected cortical pyramidal cells that physiological measurements could not be made. The speed and strength of the expression of transgenes encoded by VSV depends upon the gene's genomic position (van den Pol et al., 2009; Beier et al., 2011) . Genes in the first position are expressed the most highly, with a decrease in the level of expression in positions more 3 within the viral plus strand.", "Genes in the first position are expressed the most highly, with a decrease in the level of expression in positions more 3 within the viral plus strand. When GFP was inserted into the first position of VSV, GFP fluorescence was first detectable at approximately 1 hpi in cultured cells (van den Pol et al., 2009) . In order to quantify the relative expression of a fluorescent protein in the first genomic position in neurons, rat hippocampal slices were infected with a replication-incompetent rVSV that expresses mCherry (rVSV G, Figures 1A,B) .", "In order to quantify the relative expression of a fluorescent protein in the first genomic position in neurons, rat hippocampal slices were infected with a replication-incompetent rVSV that expresses mCherry (rVSV G, Figures 1A,B) . This was a G virus which had the RABV-G supplied in trans during the preparation of the virus stock [referred to as rVSV G(RABV-G)]. Average fluorescence intensity of the infected cells was measured every hour over the course of 18 h. By 4 hpi at 37 • C, red fluorescence was clearly visible, and reached maximal levels by approximately 14 hpi (N = 3, Figure 3 ).", "Average fluorescence intensity of the infected cells was measured every hour over the course of 18 h. By 4 hpi at 37 • C, red fluorescence was clearly visible, and reached maximal levels by approximately 14 hpi (N = 3, Figure 3 ). Similar results were obtained with a virus encoding GFP in the first genomic position rather than mCherry (i.e., Figure 1B ) (N = 3). We previously demonstrated that rVSV(RABV-G) could be taken up retrogradely by neurons (Beier et al., 2011) , but these experiments did not distinguish between direct axonal uptake of the initial inoculum vs. retrograde transsynaptic transmission following viral replication.", "We previously demonstrated that rVSV(RABV-G) could be taken up retrogradely by neurons (Beier et al., 2011) , but these experiments did not distinguish between direct axonal uptake of the initial inoculum vs. retrograde transsynaptic transmission following viral replication. To distinguish between these two mechanisms and to extend the previous analyses, we conducted further experiments in the mammalian visual system (Figures 4A-G) . As visual cortex area 1 (V1) does not receive direct projections from retinal ganglion cells (RGCs), but rather receives secondary input from RGCs via the lateral geniculate nucleus (LGN), infection of RGCs from injection of V1 would demonstrate retrograde transmission from cells which supported at least one round of viral replication.", "As visual cortex area 1 (V1) does not receive direct projections from retinal ganglion cells (RGCs), but rather receives secondary input from RGCs via the lateral geniculate nucleus (LGN), infection of RGCs from injection of V1 would demonstrate retrograde transmission from cells which supported at least one round of viral replication. Following a V1 injection with rVSV(RABV-G), GFP-positive RGCs were observed in the retina by 3 dpi (N = 3; Figure 4G ). Importantly, viral labeling in the brain was restricted to primary and secondary projection areas, even at 7 dpi.", "Importantly, viral labeling in the brain was restricted to primary and secondary projection areas, even at 7 dpi. These included the LGN ( Figure 4D ) and the hypothalamus (Figure 4E) , two areas known to project directly to V1 (Kandel, 2000) . Selective labeling was observed in other areas, such as cortical areas surrounding V1 (Figure 4C) , which project directly to V1, and also in the superior colliculus (SC) stratum griseum centrale, which projects to the LGN ( Figure 4F) .", "Selective labeling was observed in other areas, such as cortical areas surrounding V1 (Figure 4C) , which project directly to V1, and also in the superior colliculus (SC) stratum griseum centrale, which projects to the LGN ( Figure 4F) . Labeling was also observed in the nucleus basalis, which projects to the cortex, as well as many components of the basal ganglia circuit, which provide input to the thalamus [such as the caudate-putamen (CP), globus pallidus (GP), and the subthalamic nucleus (STn)]. The amygdala, which projects to the hypothalamus, was also labeled.", "The amygdala, which projects to the hypothalamus, was also labeled. Consistent with a lack of widespread viral transmission, animals did not exhibit signs of disease at 7 dpi. These data show that rVSV(RABV-G) can spread in a retrograde direction from the injection site, but do not address whether the virus can spread exclusively in the retrograde direction.", "These data show that rVSV(RABV-G) can spread in a retrograde direction from the injection site, but do not address whether the virus can spread exclusively in the retrograde direction. Directional transsynaptic specificity can only be definitively addressed using a unidirectional circuit. We therefore turned to the primary motor cortex (M1) to CP connection, in which neurons project from the cortex to the CP, but not in the other direction ( Figure 4H ) (Beier et al., 2011) .", "We therefore turned to the primary motor cortex (M1) to CP connection, in which neurons project from the cortex to the CP, but not in the other direction ( Figure 4H ) (Beier et al., 2011) . Injections of rVSV(RABV-G) into M1 should not label neurons in the CP if the virus can only label cells across synapses in the retrograde direction. Indeed, at 2 dpi, areas directly projecting to the injection site, including the contralateral cortex, were labeled ( Figure 4I ).", "Indeed, at 2 dpi, areas directly projecting to the injection site, including the contralateral cortex, were labeled ( Figure 4I ). Only axons from cortical cells were observed in the CP, with no GFP-labeled cell bodies present in the CP (Figure 4J) , consistent with lack of anterograde transsynaptic spread. By 3 dpi, a small number of medium spiny neurons (MSNs) in the CP were observed, likely via secondary spread from initially infected thalamic or GP neurons (data not shown).", "By 3 dpi, a small number of medium spiny neurons (MSNs) in the CP were observed, likely via secondary spread from initially infected thalamic or GP neurons (data not shown). A particular advantage of retrograde viral tracers is the ability to label CNS neurons projecting to peripheral sites. This has been a powerful application of both RABV and PRV (Ugolini et al., 1989; Standish et al., 1994) .", "This has been a powerful application of both RABV and PRV (Ugolini et al., 1989; Standish et al., 1994) . To test if rVSV(RABV-G) could also perform this function, we examined the innervation of the dura surface by neurons of the trigeminal ganglion, a neuronal circuit thought to be involved in migraine headaches (Penfield and McNaughton, 1940; Mayberg et al., 1984) . These neurons have axons, but not canonical dendrites, and send projections into the spinal cord and brainstem.", "These neurons have axons, but not canonical dendrites, and send projections into the spinal cord and brainstem. Therefore, the only way trigeminal neurons could become labeled from viral application to the dura is through retrograde uptake of the virus. We applied rVSV(RABV-G) to the intact dura mater and analyzed the dura, trigeminal ganglion, and CNS for labeling ( Figure 4K ).", "We applied rVSV(RABV-G) to the intact dura mater and analyzed the dura, trigeminal ganglion, and CNS for labeling ( Figure 4K ). At the earliest time point examined, 3 dpi, we observed axons traveling along the dura, but little other evidence of infection ( Figure 4L) . No labeled neuronal cell bodies on the dura were observed, consistent with the lack of neurons on this surface.", "No labeled neuronal cell bodies on the dura were observed, consistent with the lack of neurons on this surface. In contrast, we did find labeled cell bodies in the trigeminal ganglion ( Figure 4M ). No infection was seen in the CNS, even at 4 dpi, consistent with the lack of inputs from the brain into the trigeminal ganglion (N = 4 animals).", "No infection was seen in the CNS, even at 4 dpi, consistent with the lack of inputs from the brain into the trigeminal ganglion (N = 4 animals). To further characterize patterns and kinetics of viral transmission and directional specificity of transsynaptic spread, injections of rVSV(RABV-G) were made into the CP ( Figure 5A ). In order to determine which cells were labeled by direct uptake of virus in the inoculum, a separate set of animals were injected into the CP with the replication-incompetent rVSV G(RABV-G) (N = 3 animals, analyzed 3 dpi).", "In order to determine which cells were labeled by direct uptake of virus in the inoculum, a separate set of animals were injected into the CP with the replication-incompetent rVSV G(RABV-G) (N = 3 animals, analyzed 3 dpi). Cells labeled by rVSV G(RABV-G) were observed in the CP, GP, substantia nigra (SN), thalamus, and layers 3 and 5 of the cortex, consistent with infection at the axon terminal and retrograde labeling of cell bodies of neurons known to project directly to the CP ( Figure 5C ) (Albin et al., 1995) . Areas labeled by CP injection are indicated in Figure 5B .", "Areas labeled by CP injection are indicated in Figure 5B . The patterns of spread for the replication-competent rVSV(RABV-G) were characterized over the course of 1-5 dpi ( Figures 5D-H) . During this interval, progressively more cells in infected regions were labeled by rVSV(RABV-G), including within the CP, nucleus basalis, cortex, and GP (listed in Figure 5B ).", "During this interval, progressively more cells in infected regions were labeled by rVSV(RABV-G), including within the CP, nucleus basalis, cortex, and GP (listed in Figure 5B ). In addition, more cortical cells were labeled in clusters near cortical pyramidal neurons, both ipsilateral and contralateral to the injected side, including neurogliaform cells (data not shown). These data are in contrast to those observed following infection with an anterograde transsynaptic tracing virus, such as rVSV with its own G gene, rVSV(VSV-G) (Figure 5B) .", "These data are in contrast to those observed following infection with an anterograde transsynaptic tracing virus, such as rVSV with its own G gene, rVSV(VSV-G) (Figure 5B) . At 3 dpi following rVSV(VSV-G) injection into the CP, the cerebral cortex was not labeled, but regions receiving projections from the CP, such as the STn, GP, and SN, were labeled (Beier et al., 2011) . In order to investigate other areas for evidence of cell-to-cell retrograde transsynaptic spread, the nucleus basalis was examined following infection of the CP with replication-competent rVSV(RABV-G).", "In order to investigate other areas for evidence of cell-to-cell retrograde transsynaptic spread, the nucleus basalis was examined following infection of the CP with replication-competent rVSV(RABV-G). The nucleus basalis was labeled by 2 dpi (Figures 5E-H) , consistent with at least a single transsynaptic jump, as this area does not directly project to the CP. The virus appeared to travel transsynaptically at the rate of roughly 1 synapse per day, as evidenced by the lack of labeled neurogliaform cells in the cortex, and lack of neurons in the nucleus basalis at 1 dpi, and label appearing in these cell types/areas at 2 dpi, as previously observed (Beier et al., 2011) .", "The virus appeared to travel transsynaptically at the rate of roughly 1 synapse per day, as evidenced by the lack of labeled neurogliaform cells in the cortex, and lack of neurons in the nucleus basalis at 1 dpi, and label appearing in these cell types/areas at 2 dpi, as previously observed (Beier et al., 2011) . Labeling remained well-restricted to the expected corticostriatal circuits at 5 dpi, suggesting that viral spread becomes less efficient after crossing one or two connections, consistent with injections into V1 (Figure 4) . While glial cells can be infected and were observed near the injection site (van den Pol et al., 2002; Chauhan et al., 2010) , infected glial cells away from the injection site generally were not observed.", "While glial cells can be infected and were observed near the injection site (van den Pol et al., 2002; Chauhan et al., 2010) , infected glial cells away from the injection site generally were not observed. One advantage of having both anterograde and retrograde forms of the same virus is that they can be used in parallel, or in tandem, to trace circuitry to and from a single or multiple sites of injection, with each virus having similar kinetics of spread and gene expression. In fact, if different fluorophores are used in different viruses, e.g., rVSV(VSV-G) and rVSV(RABV-G), then the viruses can be co-injected into the same site and their transmission can be traced independently ( Figure 6A ).", "In fact, if different fluorophores are used in different viruses, e.g., rVSV(VSV-G) and rVSV(RABV-G), then the viruses can be co-injected into the same site and their transmission can be traced independently ( Figure 6A ). This is most straightforward if there are no cells at the injection site that are initially infected by both viruses. Co-infected cells can be easily detected, as they would express both fluorescent proteins shortly after injection.", "Co-infected cells can be easily detected, as they would express both fluorescent proteins shortly after injection. In order to determine whether two viruses would allow simultaneous anterograde and retrograde transsynaptic tracing from a single injection site, a rVSV(VSV-G) expressing Venus and a rVSV(RABV-G) expressing mCherry were injected individually (Figures 6B-D) or co-injected (Figures 6E-G) into the motor cortex, and brains were examined 3 dpi. The pattern of labeling from the co-injected brains was equivalent to the patterns observed when each virus was injected individually: rVSV(VSV-G) was observed to infect neurons in the cortex, CP, and downstream nuclei, whereas the rVSV(RABV-G) was not observed to infect neurons in the CP, but rather in the thalamus and nucleus basalis (N = 4).", "The pattern of labeling from the co-injected brains was equivalent to the patterns observed when each virus was injected individually: rVSV(VSV-G) was observed to infect neurons in the cortex, CP, and downstream nuclei, whereas the rVSV(RABV-G) was not observed to infect neurons in the CP, but rather in the thalamus and nucleus basalis (N = 4). The initial co-infection rate is dependent upon The presence or absence of labeling is indicated by (+) and (−), respectively. The extent of labeling is indicated by the number of (+).", "The extent of labeling is indicated by the number of (+). Some animals were infected with G viruses to determine which areas were labeled by direct uptake of the virions, rather than by replication and transmission. These were sacrificed at 3 dpi.", "These were sacrificed at 3 dpi. (C) Parasaggital section of a brain infected with VSV[greek delta]G(RABV-G). The injection site is marked by a red arrow.", "The injection site is marked by a red arrow. Several areas that project directly to the CP were labeled due to direct uptake of the virions, including the cortex, thalamus, and GP (arrowheads), 3 dpi. the dose of the initial inocula.", "the dose of the initial inocula. When injecting 3 × 10 3 focus forming units (ffu) rVSV(VSV-G) and 3 × 10 4 ffu rVSV(RABV-G), no co-infection was observed at the injection site. Thus, co-infection of the same brain region, without co-infection of the same cells,\n\ndoes not alter the spreading behavior of either rVSV(VSV-G) or rVSV(RABV-G).", "Thus, co-infection of the same brain region, without co-infection of the same cells,\n\ndoes not alter the spreading behavior of either rVSV(VSV-G) or rVSV(RABV-G). One example of how this dual retrograde and anterograde transsynaptic tracing system can be used is to determine if three Frontiers in Neural Circuits February 2013 | Volume 7 | Article 11 | 8 distinct regions are connected and the directionality of any connections. For example, the anterograde transsynaptic virus can be injected into one region, the retrograde into another, and a third region can then be examined for evidence of labeling by either or both viruses (e.g., Figure 6H ).", "For example, the anterograde transsynaptic virus can be injected into one region, the retrograde into another, and a third region can then be examined for evidence of labeling by either or both viruses (e.g., Figure 6H ). To test this possibility, rVSV(VSV-G) was injected into the motor cortex, rVSV(RABV-G) was injected into the substantia nigra pars reticulara (SNr), and animals were sacrificed at 3 dpi. We observed that cells were singly labeled, either with Venus [rVSV(VSV-G)] or with mCherry [rVSV(RABV-G)], and were located largely in different regions of the CP (Figures 6I,J) (N = 3) .", "We observed that cells were singly labeled, either with Venus [rVSV(VSV-G)] or with mCherry [rVSV(RABV-G)], and were located largely in different regions of the CP (Figures 6I,J) (N = 3) . These results suggest that the anterograde connections from the cells infected with rVSV(VSV-G) in the M1 were with CP MSNs that did not project to the region of the SNr injected with rVSV(RABV-G) (N = 3 animals). In addition to polysynaptic tracing, VSV can be modified to trace circuits monosynaptically (Beier et al., 2011) .", "In addition to polysynaptic tracing, VSV can be modified to trace circuits monosynaptically (Beier et al., 2011) . With RABV, this was achieved in vivo by first infecting with an adeno-associated virus (AAV) expressing TVA, a receptor for an avian retrovirus, and RABV-G (Wall et al., 2010) . This was followed 3 weeks later by infection with a G RABV with an EnvA/RABV-G chimeric glycoprotein on the virion surface (Wickersham et al., 2007b) , which allowed infection specifically of the cells expressing TVA.", "This was followed 3 weeks later by infection with a G RABV with an EnvA/RABV-G chimeric glycoprotein on the virion surface (Wickersham et al., 2007b) , which allowed infection specifically of the cells expressing TVA. A similar strategy was used to test rVSV's ability to monosynaptically trace retrogradely connected neurons in vivo. Inputs to choline acetyltransferase (ChAT)-expressing neurons in the striatum were used for this test.", "Inputs to choline acetyltransferase (ChAT)-expressing neurons in the striatum were used for this test. These neurons primarily receive input from the cortex and the thalamus (Thomas et al., 2000; Bloomfield et al., 2007) (Figure 7A) . In order to mark this population, we crossed ChAT-Cre mice to Ai9 mice, which express tdTomato in cells with a Cre expression history (Madisen et al., 2010) .", "In order to mark this population, we crossed ChAT-Cre mice to Ai9 mice, which express tdTomato in cells with a Cre expression history (Madisen et al., 2010) . Six-week-old mice from this cross were injected in the CP with two AAV vectors: one expressing a Cre-conditional (\"floxed\") TVA-mCherry fusion protein, and another expressing a floxed RABV-G. Two weeks later, the mice were injected in the same coordinates with rVSV G with the EnvA/RABV-G chimeric glycoprotein on the virion surface [rVSV G(A/RG)] (Beier et al., 2011) . Cells successfully infected with these two AAV vectors could host infection by a rVSV and should be able to produce rVSV virions with RABV-G on the surface.", "Cells successfully infected with these two AAV vectors could host infection by a rVSV and should be able to produce rVSV virions with RABV-G on the surface. Such starter cells should also express tdTomato and GFP. If rVSV were to be produced, and if it were to transmit across the synapse retrogradely, cortical and thalamic neurons should be labeled by GFP.", "If rVSV were to be produced, and if it were to transmit across the synapse retrogradely, cortical and thalamic neurons should be labeled by GFP. Mice injected with these AAV and rVSV viruses were sacrificed 5 days after rVSV infection, and brains analyzed for fluorescence. As expected for starter cells, some neurons in the CP expressed both tdTomato and GFP (Figure 7B) .", "As expected for starter cells, some neurons in the CP expressed both tdTomato and GFP (Figure 7B) . Outside of the CP, small numbers of GFP+ neurons that were not mCherry+ were observed in the cortex (Figures 7C,D) and thalamus (Figure 7E) , consistent with retrograde spread. Control animals not expressing Cre, or not injected with AAV encoding RABV-G, did not label cells in the cortex or thalamus (N = 3 for both controls and experimental condition).", "Control animals not expressing Cre, or not injected with AAV encoding RABV-G, did not label cells in the cortex or thalamus (N = 3 for both controls and experimental condition). Here, we report on the use of rVSV as a retrograde transsynaptic tracer for CNS circuitry. VSV can be modified to encode the RABV-G protein in the viral genome, allowing the virus to replicate and transmit across multiple synaptically connected cells, i.e., as a polysynaptic tracer.", "VSV can be modified to encode the RABV-G protein in the viral genome, allowing the virus to replicate and transmit across multiple synaptically connected cells, i.e., as a polysynaptic tracer. Alternatively, if the virus has the G gene deleted from its genome and RABV-G is provided in trans, it behaves as a monosynaptic tracer (Beier et al., 2011) . Although it has been known for many years that RABV travels retrogradely among neurons (Astic et al., 1993; Ugolini, 1995; Kelly and Strick, 2003) , and pseudotyping lentiviruses with RABV-G is sufficient for axonal transport (Mazarakis et al., 2001) , the retrograde transmission specificity among neurons had not been clearly shown to be a property of the G protein itself, as it might have been due to other viral proteins in addition to, or instead of, the viral G protein.", "Although it has been known for many years that RABV travels retrogradely among neurons (Astic et al., 1993; Ugolini, 1995; Kelly and Strick, 2003) , and pseudotyping lentiviruses with RABV-G is sufficient for axonal transport (Mazarakis et al., 2001) , the retrograde transmission specificity among neurons had not been clearly shown to be a property of the G protein itself, as it might have been due to other viral proteins in addition to, or instead of, the viral G protein. Since native VSV does not have these retrograde transsynaptic properties (van den Pol et al., 2002; Beier et al., 2011) , and the only alteration to the VSV genome was the substitution of the VSV G gene with the G gene of RABV, it is clear that the RABV glycoprotein is responsible for retrograde direction of viral transmission across synapses, at least in the case of rVSV. The early onset of gene expression from VSV relative to RABV (one hour vs. multiple hours) makes it beneficial in experimental paradigms in which the experiment needs to be done within a narrow window of time, such as tissue slices and explants.", "The early onset of gene expression from VSV relative to RABV (one hour vs. multiple hours) makes it beneficial in experimental paradigms in which the experiment needs to be done within a narrow window of time, such as tissue slices and explants. In addition, more than one transgene can be encoded in the viral genome without the need of a 2A or IRES element. The use of the first position of the genome enhances the expression level of the transgene inserted at that location, since VSV (and RABV) express genes in a transcriptional gradient; therefore, the first gene is the most highly transcribed (Knipe, 2007) .", "The use of the first position of the genome enhances the expression level of the transgene inserted at that location, since VSV (and RABV) express genes in a transcriptional gradient; therefore, the first gene is the most highly transcribed (Knipe, 2007) . This leads to rational predictions of expression levels so that one can choose the position of insertion of a transgene, or transgenes, according to this gradient and the desired level of expression. The size of the viral capsid is apparently not rigid, allowing for the inclusion of genomes that are substantially larger than the native genome, unlike the rigid capacity for some other viral vectors, such as AAV Yan et al., 2000) .", "The size of the viral capsid is apparently not rigid, allowing for the inclusion of genomes that are substantially larger than the native genome, unlike the rigid capacity for some other viral vectors, such as AAV Yan et al., 2000) . The fact that VSV can be made to spread anterogradely (Beier et al., 2011) or retrogradely across synapses with the change of a single gene affords several advantages over viral tracers that heretofore have not shown such flexibility in the directionality of tracing. In addition to the obvious application of tracing anterograde connections, combinations can be made to exploit the different forms of the virus.", "In addition to the obvious application of tracing anterograde connections, combinations can be made to exploit the different forms of the virus. One example that employs the simultaneous infection with an anterograde and retrograde form of VSV is demonstrated in Figure 6 . This experiment was designed to address whether the anterograde projections from the cortex to the CP would label the same brain regions as were labeled by a retrograde virus injected into the SN.", "This experiment was designed to address whether the anterograde projections from the cortex to the CP would label the same brain regions as were labeled by a retrograde virus injected into the SN. Although a block of superinfection by the virus may preclude infection of the same cell with multiple rVSVs, adjacent cells could still become labeled by different viruses (Whitaker-Dowling et al., 1983) . The observed results could be due to a preferential labeling by the anterograde transsynaptic virus of indirect pathway MSNs in this experiment, which then synapse onto the GP, thereby reflecting a viral bias.", "The observed results could be due to a preferential labeling by the anterograde transsynaptic virus of indirect pathway MSNs in this experiment, which then synapse onto the GP, thereby reflecting a viral bias. Alternatively, it could indicate that the cortical neurons in the injected region largely do not label the MSNs that project to the area of the SN injected with the retrograde virus. One further possibility is that too little virus was used to observe co-labeling of a given region.", "One further possibility is that too little virus was used to observe co-labeling of a given region. However, given the density of infection (i.e., Figures 6I,J) , the latter possibility seems unlikely. Additionally, the spread of the polysynaptic rVSV(RABV-G) appears to attenuate with increasing numbers of synapses crossed, permitting an analysis of more restricted viral spread.", "Additionally, the spread of the polysynaptic rVSV(RABV-G) appears to attenuate with increasing numbers of synapses crossed, permitting an analysis of more restricted viral spread. This is quite fortuitous, as if spread were to continue, it would lead to widespread infection and lethality. In addition, reconstruction of connectivity would be more difficult.", "In addition, reconstruction of connectivity would be more difficult. This reduced efficiency appears to also hold for the monosynaptic form of VSV complemented with RABV-G, as the efficiency of transmission appeared lower than the comparable experiment with RABV (Watabe-Uchida et al., 2012) . This is likely due to viral attenuation when VSV-G is replaced with RABV-G.\n\nWe were attracted to the use of VSV as a viral tracer due to its long track record as a safe, replication-competent laboratory agent.", "This is likely due to viral attenuation when VSV-G is replaced with RABV-G.\n\nWe were attracted to the use of VSV as a viral tracer due to its long track record as a safe, replication-competent laboratory agent. Laboratory workers using VSV have not contracted any diseases, and natural VSV infections among human populations in Central America and the southwestern United States (Rodríguez, 2002) occur without evident pathology (Johnson et al., 1966; Brody et al., 1967) . VSV was thus an attractive candidate for its use as a polysynaptic tracer for CNS studies, which requires an ability to replicate through multiple transmission cycles.", "VSV was thus an attractive candidate for its use as a polysynaptic tracer for CNS studies, which requires an ability to replicate through multiple transmission cycles. Both replicationcompetent and incompetent forms of VSV are in use under Biosafety Level 2 containment. Replication-competent RABV is Biosafety Level 3, due to the fact that infection with replicationcompetent RABV is almost always fatal to humans and in mice when infected intracerebrally (Smith, 1981; Knipe, 2007) .", "Replication-competent RABV is Biosafety Level 3, due to the fact that infection with replicationcompetent RABV is almost always fatal to humans and in mice when infected intracerebrally (Smith, 1981; Knipe, 2007) . Differences in pathogenicity between VSV and RABV are likely due to the ability of RABV to evade the innate immune system, particularly interferon (Hangartner et al., 2006; Junt et al., 2007; Lyles and Rupprecht, 2007; Rieder and Conzelmann, 2009; Iannacone et al., 2010) . VSV infection efficiently triggers an interferon response, and it has not evolved a method of escape from this response, unlike RABV (Brzózka et al., 2006) .", "VSV infection efficiently triggers an interferon response, and it has not evolved a method of escape from this response, unlike RABV (Brzózka et al., 2006) . In fact, VSV is being pursued as a vaccine for other viruses, including RABV (Lichty et al., 2004; Publicover et al., 2004; Kapadia et al., 2005; Schwartz et al., 2007; Iyer et al., 2009; Geisbert and Feldmann, 2011) . VSV does not typically spread beyond the initially infected site in the periphery (Kramer et al., 1983; Vogel and Fertsch, 1987) .", "VSV does not typically spread beyond the initially infected site in the periphery (Kramer et al., 1983; Vogel and Fertsch, 1987) . This likely is the cause of the minor or absent symptoms in humans and animals infected in nature. Polysynaptic VSV vectors are thus predicted to be much safer than polysynaptic RABV vectors.", "Polysynaptic VSV vectors are thus predicted to be much safer than polysynaptic RABV vectors. We have tested this prediction by injecting a series of mice in the footpads and hind leg muscles with rVSV(RABV-G), with the result that no injected animals showed any evidence of morbidity or mortality (Beier, Goz et al., in preparation) . While safer for laboratory workers than RABV, the main drawback to using VSV is its rapid cellular toxicity (van den Pol et al., 2009; Beier et al., 2011) .", "While safer for laboratory workers than RABV, the main drawback to using VSV is its rapid cellular toxicity (van den Pol et al., 2009; Beier et al., 2011) . Toxicity is due to suppression of cellular transcription and a block in the export of cellular RNAs from the nucleus to the cytoplasm (Black and Lyles, 1992; Her et al., 1997; Ahmed and Lyles, 1998; Petersen et al., 2000; von Kobbe et al., 2000) , as well as inhibition of the translation of cellular mRNAs (Francoeur et al., 1987; Jayakar et al., 2000; Kopecky et al., Frontiers in Neural Circuits February 2013 | Volume 7 | Article 11 | 10 2001). VSV is much quicker to enact its gene expression program than is RABV, such that cells suffer the toxic effects more quickly than after RABV infection.", "VSV is much quicker to enact its gene expression program than is RABV, such that cells suffer the toxic effects more quickly than after RABV infection. One aspect of VSV that can be exploited in the future to ameliorate the speed of toxicity is the use of VSV mutants and variants. One such mutant is the M51R, which permitted us to conduct physiological analyses of pre-and post-synaptic cells (Beier et al., 2011) .", "One such mutant is the M51R, which permitted us to conduct physiological analyses of pre-and post-synaptic cells (Beier et al., 2011) . We are in the process of examining the transmission properties of this mutant in vivo, as well as the effects of other mutations or viral variants on prolonging the health of neurons after infection. rVSV vectors can be used to study the connectivity of neuronal circuitry.", "rVSV vectors can be used to study the connectivity of neuronal circuitry. In addition to combinations of replication-competent forms of VSV, the replication-incompetent, monosynaptic forms of the virus can be easily combined, without the need to change viruses (Beier et al., 2011) . This allows a straightforward way to study both the projections into, and out from, a genetically defined cell population.", "This allows a straightforward way to study both the projections into, and out from, a genetically defined cell population. This can be done with the same viral genome, with the only change needed being the glycoprotein, for the selection of the direction of transmission. This flexibility of VSV makes it a powerful, multi-application vector for studying connectivity in the CNS.", "This flexibility of VSV makes it a powerful, multi-application vector for studying connectivity in the CNS. All rVSV clones were cloned from the rVSV G backbone (Chandran et al., 2005) . mCherry, Kusabira orange, Venus, and CFP were cloned into the first (GFP) position using XhoI and MscI sites, and VSV-G (a gift from Richard Mulligan, Harvard Medical School, Boston, MA) and RABV-G (a gift from Ed Callaway, Salk Institute, San Diego, CA) were cloned into the fifth (G) position using the MluI and NotI restriction sites.", "mCherry, Kusabira orange, Venus, and CFP were cloned into the first (GFP) position using XhoI and MscI sites, and VSV-G (a gift from Richard Mulligan, Harvard Medical School, Boston, MA) and RABV-G (a gift from Ed Callaway, Salk Institute, San Diego, CA) were cloned into the fifth (G) position using the MluI and NotI restriction sites. Genes for fluorescent proteins were obtained from Clontech. Viruses were rescued as previously described (Whelan et al., 1995) .", "Viruses were rescued as previously described (Whelan et al., 1995) . At 95% confluency, eight 10 cm plates of BSR cells were infected at an MOI of 0.01. Viral supernatants were collected at 24-h time intervals and ultracentrifuged at 21,000 RPM using a SW28 rotor and resuspended in 0.2% of the original volume.", "Viral supernatants were collected at 24-h time intervals and ultracentrifuged at 21,000 RPM using a SW28 rotor and resuspended in 0.2% of the original volume. For titering, concentrated viral stocks were applied in a dilution series to 100% confluent BSR cells and plates were examined at 12 hpi. Viral stocks were stored at −80 • C.\n\nFor G viruses, 293T cells were transfected with PEI (Ehrhardt et al., 2006) at 70% confluency on 10 cm dishes with 5 µg of pCAG-RABV-G. Twenty-four hours post-infection, the cells were infected at an MOI of 0.01 with rVSV G expressing either GFP or mCherry.", "Viral stocks were stored at −80 • C.\n\nFor G viruses, 293T cells were transfected with PEI (Ehrhardt et al., 2006) at 70% confluency on 10 cm dishes with 5 µg of pCAG-RABV-G. Twenty-four hours post-infection, the cells were infected at an MOI of 0.01 with rVSV G expressing either GFP or mCherry. Viral supernatants were collected for the subsequent 4 days at 24 h intervals. Virus preparations are now available from the Salk GT3 viral core ( All plasmids are available from Addgene (\n\nAAV-FLEx-RABV-G and AAV-FLEx-TVA-mCherry plasmids originated from the Lab of Naoshige Uchida (Watabe-Uchida et al., 2012) , and virus stocks were generous gifts from Brad Lowell, Harvard Medical School.", "Virus preparations are now available from the Salk GT3 viral core ( All plasmids are available from Addgene (\n\nAAV-FLEx-RABV-G and AAV-FLEx-TVA-mCherry plasmids originated from the Lab of Naoshige Uchida (Watabe-Uchida et al., 2012) , and virus stocks were generous gifts from Brad Lowell, Harvard Medical School. ChAT-Cre (B6;129S6-Chat tm1(cre)Lowl /J) and Ai9 (B6.Cg-Gt(ROSA)26Sor<tm9(CAG-tdTomato)Hze>/J) mice were obtained from the Jackson Laboratory (Madisen et al., 2010) . Eight-week-old CD-1 mice were injected using pulled capillary microdispensers (Drummond Scientific, Cat.", "Eight-week-old CD-1 mice were injected using pulled capillary microdispensers (Drummond Scientific, Cat. No: 5-000-2005) , using coordinates from The Mouse Brain in Stereotaxic Coordinates (Franklin and Paxinos, 1997) . Injection coordinates (in mm) used were: For multi-color analysis (Figures 1C,D) , 3 × 10 9 ffu/mL rVSV was injected into various regions.", "Injection coordinates (in mm) used were: For multi-color analysis (Figures 1C,D) , 3 × 10 9 ffu/mL rVSV was injected into various regions. For CP injections, 100 nL of rVSV(RABV-G) or rVSV(VSV-G) at 3 × 10 7 ffu/mL was injected at a rate of 100 nL/min. For the replication-incompetent viruses, 100 nL of 1 × 10 7 ffu/mL rVSV G(RABV-G) or rVSV G (VSV-G) was injected.", "For the replication-incompetent viruses, 100 nL of 1 × 10 7 ffu/mL rVSV G(RABV-G) or rVSV G (VSV-G) was injected. In the motor cortex, 100 nL of 1 × 10 7 ffu/mL rVSV(RABV-G) was injected, and mice harvested 2 dpi. For V1 injections, 100 nL of 3 × 10 10 ffu/mL rVSV(RABV-G) was injected, and mice were examined 3 or 7 dpi.", "For V1 injections, 100 nL of 3 × 10 10 ffu/mL rVSV(RABV-G) was injected, and mice were examined 3 or 7 dpi. For infections of the dura mater, 1 µL of 3 × 10 10 ffu/mL rVSV(RABV-G) was applied to the surface of the dura. The virus was allowed to absorb, and the surface was subsequently covered in bone wax, and the wound sutured.", "The virus was allowed to absorb, and the surface was subsequently covered in bone wax, and the wound sutured. For co-injections of virus into the same animal, 100 nL of a combination of 3 × 10 7 ffu/mL rVSV(VSV-G) and 3 × 10 8 ffu/mL rVSV(RABV-G) were co-injected into the motor cortex, and brains examined 3 dpi. For injections of the viruses into different regions, 100 nL of 3 × 10 7 ffu/mL rVSV(VSV-G) was injected into M1, and 100 nL of 3 × 10 8 ffu/mL rVSV(RABV-G) into the SNr, and brains examined 3 dpi.", "For injections of the viruses into different regions, 100 nL of 3 × 10 7 ffu/mL rVSV(VSV-G) was injected into M1, and 100 nL of 3 × 10 8 ffu/mL rVSV(RABV-G) into the SNr, and brains examined 3 dpi. A lower titer of rVSV(VSV-G) was used, as rVSV(RABV-G) is attenuated. All mouse work was conducted in biosafety containment level 2 conditions and was approved by the Longwood Medical Area Institutional Animal Care and Use Committee.", "All mouse work was conducted in biosafety containment level 2 conditions and was approved by the Longwood Medical Area Institutional Animal Care and Use Committee. Recordings were made from cortical pyramidal neurons in slices taken from postnatal day 12-18 mice, inoculated in the CP 12-18 h prior with rVSV(RABV-G). Coronal slices (300 µm thick) were cut in ice-cold external solution containing (in mM): 110 choline, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 2.5 KCl, 7 MgCl 2 , 0.5 CaCl 2 , 25 glucose, 11.6 Na-ascorbate, and 3.1 Na-pyruvate, bubbled with 95% O 2 and 5% CO 2 .", "Coronal slices (300 µm thick) were cut in ice-cold external solution containing (in mM): 110 choline, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 2.5 KCl, 7 MgCl 2 , 0.5 CaCl 2 , 25 glucose, 11.6 Na-ascorbate, and 3.1 Na-pyruvate, bubbled with 95% O 2 and 5% CO 2 . Slices were then transferred to artificial cerebrospinal fluid (ACSF) containing (in mM): 127 NaCl, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 2.5 KCl, 1 MgCl 2 , 2 CaCl 2 , and 25 glucose, bubbled with 95% O 2 and 5% CO 2 . After an incubation period Frontiers in Neural Circuits February 2013 | Volume 7 | Article 11 | 11 of 30-40 min at 34 • C, slices were stored at room temperature.", "After an incubation period Frontiers in Neural Circuits February 2013 | Volume 7 | Article 11 | 11 of 30-40 min at 34 • C, slices were stored at room temperature. All experiments were conducted at room temperature (25 • C). In all experiments, 50 µM picrotoxin, 10 µM 2,3-Dioxo-6-nitro-1, 2, 3, 4 -tetrahydrobenzo [f]quinoxaline -7-sulfonamide (NBQX), and 10 µM 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) were present in the ACSF to block GABAA/C, AMPA, and NMDA receptor-mediated transmission, respectively.", "In all experiments, 50 µM picrotoxin, 10 µM 2,3-Dioxo-6-nitro-1, 2, 3, 4 -tetrahydrobenzo [f]quinoxaline -7-sulfonamide (NBQX), and 10 µM 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) were present in the ACSF to block GABAA/C, AMPA, and NMDA receptor-mediated transmission, respectively. All chemicals were from Sigma or Tocris. Whole-cell recordings were obtained from infected and uninfected deep layer cortical pyramidal neurons identified with video-IR/DIC and GFP fluorescence was detected using epifluorescence illumination.", "Whole-cell recordings were obtained from infected and uninfected deep layer cortical pyramidal neurons identified with video-IR/DIC and GFP fluorescence was detected using epifluorescence illumination. With the deep layers of the cortex, 2-photon laser scanning microscopy (2PLSM) was used to confirm the cell types based on morphology. Deep layer pyramidal neurons had large cell bodies, classic pyramidal shape and dendritic spines.", "Deep layer pyramidal neurons had large cell bodies, classic pyramidal shape and dendritic spines. Glass electrodes (2-4 M ) were filled with internal solution containing (in mM): 135 KMeSO 4 , 5 KCl, 5 HEPES, 4 MgATP, 0.3 NaGTP, 10 Na 2 HPO 4 , 1 EGTA, and 0.01 Alexa Fluor-594 (to image neuronal morphology) adjusted to pH 7.4 with KOH. Current and voltage recordings were made at room temperature using a AxoPatch 200B or a Multiclamp 700B amplifier.", "Current and voltage recordings were made at room temperature using a AxoPatch 200B or a Multiclamp 700B amplifier. Data was filtered at 5 kHz and digitized at 10 kHz. Imaging and physiology data were acquired and analyzed as described previously (Carter and Sabatini, 2004) .", "Imaging and physiology data were acquired and analyzed as described previously (Carter and Sabatini, 2004) . Resting membrane potential was determined by the average of three 5-s sweeps with no injected current. Passive properties of the cell, membrane (Rm) and series resistance (Rs) and capacitance (Cm), were measured while clamping cells at −65 mV and applying voltage steps from −55 to −75 mV.", "Passive properties of the cell, membrane (Rm) and series resistance (Rs) and capacitance (Cm), were measured while clamping cells at −65 mV and applying voltage steps from −55 to −75 mV. The current-firing relationship was determined in current clamp with 1-s periods of injected current from 100 to 500 pA. The time course of viral gene expression experiments were carried out in organotypic hippocampal slice cultures prepared from postnatal day 5-7 Sprague-Dawley rats as described previously (Stoppini et al., 1991) .", "The time course of viral gene expression experiments were carried out in organotypic hippocampal slice cultures prepared from postnatal day 5-7 Sprague-Dawley rats as described previously (Stoppini et al., 1991) . Slices were infected after 7 days in vitro, and images were acquired on a two-photon microscope." ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.", "Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo.", "Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.", "CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%.", "Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] .", "However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups.", "However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation.", "For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found.", "Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .", "The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen.", "As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] .", "This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations.", "Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ).", "Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] .", "They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] .", "Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] .", "The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days.", "The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.", "Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries.", "Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] .", "Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy.", "Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline.", "The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] .", "156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] .", "In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model.", "In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%.", "The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use.", "This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as \"carrageenan\"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis.", "Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared.", "For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).", "MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].", "All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later).", "Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively.", "For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation.", "The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C.", "Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm.", "Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds.", "After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments.", "All animal experiments were carried out according to the guidelines of the \"European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes\" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna.", "C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively.", "For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.", "Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] .", "Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight.", "Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains.", "As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] .", "To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination.", "Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances.", "Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ).", "The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) .", "The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction.", "A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) .", "In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds.", "As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia.", "Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice.", "First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment.", "Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15.", "All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016).", "Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy.", "Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident.", "However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored.", "Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found.", "All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice.", "In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments.", "Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment.", "In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models.", "There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted \n\nWe investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing.", "However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination.", "Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig).", "The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells.", "For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir.", "This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms.", "Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) .", "The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] .", "Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract.", "In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice.", "The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof.", "First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day).", "(B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given.", "On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs.", "Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks.", "was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] .", "The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) .", "They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model.", "Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] .", "These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies.", "This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] .", "The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) .", "We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi.", "Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach.", "The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge.", "Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV.", "Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses.", "In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent.", "In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] .", "However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy.", "To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown).", "After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other.", "In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected.", "A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced.", "Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections.", "Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need.", "In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] .", "Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir).", "At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections.", "Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice.", "Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days.", "Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown).", "Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)" ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria.", "A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses.", "Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year.", "In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI. Text: Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans [1] . Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] .", "Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies [2] . However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50% [3] .", "Unfortunately, these infections causes are identified in less than 50% [3] . Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] .", "The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France [4] . Influenza activity generally increases during austral winter, corresponding to summer in Europe [5] . Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.", "Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere. Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner's network [6] . In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs.", "In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion.", "Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology. Several biological tools allow identifying respiratory pathogens from nasal swab.", "Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7] [8] [9] [10] . We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.", "We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012. The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.", "Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season. ILI was defined as a sudden onset of fever more than 38 degrees Celsius and cough, associated or not with other symptoms such as breathing difficulty, headache, etc. Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days.", "Every week, all GPs of the sentinel network were encouraged to collect a nasal swab from the first two patients who presented ILI since less than three days. After being tested for influenza viruses, the 994 swabs collected in 2011 and 2012 are frozen at -80°C at the university hospital center (CHU) laboratory. Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season.", "Based on the budget, a season-stratified sample of 250 swabs was randomly selected in order to describe circulating viruses including outside flu season. Random sampling was performed with Excel 1 using the anonymized surveillance database of the Cire OI. The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR.", "The sampling frame contained identification number of swab assigned by Cire OI, laboratory identification number, sex, age, date of onset of symptoms, date of swab collection and result of influenza RT-PCR. We used Respifinder 1 Smart 22 kits a multiplex RT-PCR (PathoFinder, Maastricht, The Netherlands) which can detect 22 respiratory pathogens. This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology.", "This assay is based on the multiplex ligation-dependent probe amplification (MLPA) technology. The reverse transcription and preamplification steps were performed on the epgradient Mastercycler 1 (Eppendorf) and the hybridization, ligation and detection steps on the LightCycler 1 480 system (Roche Applied Science). This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] .", "This method was chosen because of its high specificity, compared to other same methods (78% versus 33%) [3, 11] . Multiplex analysis allows for rapid production of diagnostic results. It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 .", "It thus allows highlighted the possible presence of eighteen respiratory viruses and four bacteria in one reaction by melt curve analysis: Influenza A not (H1N1 \n\nStatistical analyses were performed with Stata 1 and Excel 1 . Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year. Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012.", "Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.", "Data were collected for surveillance purpose and are totally anonymous. Among the 250 randomly-selected swabs, 26 were not available anymore as they were sent to Influenza Reference Center for confirmation and characterization of the pathogenic agent. According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR.", "According to the sensitivity of the assay two samples could be discordant results between Influenza PCR initially realized and Multiplex PCR. Thus they were deleted from the analysis: one is positive for Influenza in singleplex and negative for all tested pathogens in multiplex and one is positive for Influenza in singleplex and positive for PIV2 in multiplex. In total, 222 analyses were considered.", "In total, 222 analyses were considered. Moreover, 53 samples were negative for all analyzed respiratory pathogens (23.9%) and 169 samples had at least one detected pathogen (76.1%), finally a total of 178 pathogens was identified. During the study period, a minority of the weeks (21 i.e.", "During the study period, a minority of the weeks (21 i.e. 20%) did not include any sampled swab, mainly outside flu season. Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81].", "Patients' sex-ratio was 0.63 (86 men and 136 women) and mean age was 28.4 years [min 0; max 81]. Ten percent had less than 5 years, 24% 5-15 years, 63% 15-65 years and only 3% were 65 and older. The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) .", "The respiratory pathogens most frequently identified in ILI swabs were rhinovirus (23.4%), influenza A not H1N1 (21.2%) and influenza B (12.6%) ( Table 1) . Among the 22 respiratory pathogens tested by the multiplex, only three were not found in any analyzed sample: Parainfluenza3, Legionella pneumophila and Bordetella pertussis. Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) .", "Regarding co-infections, nine swabs revealed the presence of two viruses, among which6 involved influenza viruses (Table 2) . Analyses showed that some viruses are possibly seasonal and were circulating during a specific period of the year. They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09.", "They are detected only in summer for Human Metapneumovirus, RSV A and B, and influenza A(H1N1)pdm09. For the latter, it is specific to the studied period since the influenza A(H1N1)pdm09 virus reappeared in Réunion Island in October 2012 and was no longer circulating since late 2010. On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter.", "On the opposite, Parainfluenza 1,2 and 4 viruses were identified only in winter. For other pathogens, no specific period of detection was observed. A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) .", "A weekly description of samples was realized to study the distribution of respiratory pathogens in 2011 and 2012 (Fig 1) . Results of biological analyses were compared with data of ILI consultations declared by sentinel GPs in 2011 and 2012. We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses.", "We observed in 2011, after a first wave in June mainly due to influenza A not H1N1 virus, a second wave of ILI consultations with mainly identification of Parainfluenza viruses and not influenza viruses. In 2012, the second epidemic wave at the end of austral winter coincided with Influenza viruses and Rhinovirus circulation. Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season.", "Regarding negative swabs (Fig 2) , we observed no seasonality during the study period with a similar proportion whatever the season. This retrospective study based on a sentinel GPs network showed that not only influenza viruses are responsible for ILI consultations. Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012.", "Indeed, an important circulation of multiple pathogens was observed throughout the year, with 12 different types of pathogens identified in 2011 and 2012. Respiratory viral pathogens were present in 76.1% of samples, which is largely above results from annual influenza surveillance [12] . After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island.", "After influenza viruses, Rhinovirus and Coronavirus were the most common respiratory viruses in Réunion Island. Although samples were not taken every week, sample was representative of ILI activity and consistent with flu season. Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality.", "Nevertheless, according to the low number of samples, it is difficult to conclude about seasonality. However in our study, RSV was circulating in summer season which is hot and rainy, which is confirmed by other studies in tropical region [13] . This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI.", "This study also highlighted several co-infections, showing that concomitant the multiple etiology of ILI. Co-circulation was already observed in Réunion Island during the A(H1N1) pdm09 pandemic in addition to influenza virus, with identification of other respiratory viruses such as Rhinovirus or Coronavirus [14] . In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] .", "In mainland France, during this pandemic, circulation of major respiratory viruses was found, such as Rhinovirus, Parainfluenza, Coronavirus, Human Metapneumovirus, like in our publication [15] [16] . In our study, only 5.3% of positive swabs were co-infections whereas in two studies in Madagascar co-infections represented 27.3% and 29.4% [17] [18] . Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France.", "Despite the distance of 9,300 km between Réunion and France, the island is directly connected to Europe with four daily flights to France. These exchanges can impact respiratory pathogens circulation in southern and northern hemisphere. Results of this study can therefore be of interest to both Indian Ocean and Europe countries.", "Results of this study can therefore be of interest to both Indian Ocean and Europe countries. Among the 148 swabs initially negative for influenza because not previously tested for any other viruses, the study found an etiology for 95 swabs. In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year.", "In total, only 53 swabs, representing 24% of the sample, remained without etiology with negative multiplex PCR results all along the year. Multiple hypotheses can explain this result: a poor quality of swabs, preventing from identifying a pathogen, noninfectious causes or other pathogens not included in the multiplex PCR. However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells.", "However, we couldn't test the negative swabs for RNAse P, a marker of human cells, which could provide a modicum of assurance that the swab contained human cells. Concerning the two samples divergent for influenza identification between the multiplex and singleplex PCR, we discarded them for the analysis; one was positive in Influenza with singleplex and positive in PIV with multiplex. It could be a false positive result from singleplex.", "It could be a false positive result from singleplex. Indeed, as the multiplex PCR assay has a good sensitivity and is considered as a gold-standard, we decided to keep seven negative results for Influenza in singleplex and positive in Influenza in multiplex [7] [8] [9] [10] . No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study.", "No case of Bordetella pertussis which causes whooping cough and Legionella pneumophila which causes Legionnaires' disease was identified in this study. However, these diseases are rare in Réunion Island, around three cases of Legionnaires' disease are declared each year. A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island.", "A limit of the study is that no clinical data were available in the virological surveillance system of influenza in Réunion Island. It was impossible to compare clinical symptoms according to each pathogen and to know if there are different pathogens which cause for instance rhinitis, laryngitis or bronchitis (diseases included in ILI). A specific prospective study including clinical data might provide useful elements in the semiotics of diseases.", "A specific prospective study including clinical data might provide useful elements in the semiotics of diseases. In conclusion, this study highlighted an important circulation of multiple pathogens in Réunion Island throughout the year. It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms.", "It shows that ILI is not specific to influenza and so it is essential to have biological results in order to establish the differential diagnosis and thus explain the etiology of symptoms. For a better understanding of respiratory pathogens circulating in Réunion Island, information from this study may also be useful to practitioners who see many patients in consultation with ILI. As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI.", "As the use of multiplex RT-PCR showed its efficacy in the ILI surveillance and allowed to highlight the circulation of other viruses and bacterial causes of respiratory infections, it is now used routinely in the surveillance of ILI. Moreover, it would be interesting to repeat this study every 3 or 5 years adding clinical data to monitor the evolution of respiratory pathogens in Réunion Island over time." ]

[ "nan\n\nText: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 (COVID-19) ( com/novel-coronavirus-covid-19-portal/). According to what has been reported [1] [2] [3] , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome (SARS) caused by SARS-CoV.", "According to what has been reported [1] [2] [3] , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome (SARS) caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity [4, 5] . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin.", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV (RaTG13) was recently reported, which shared ∼96% homology with the SARS-CoV-2 [4] . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide (nt) variations (SNVs) identified across the genome [6] .", "However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide (nt) variations (SNVs) identified across the genome [6] . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV [4] , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species (bats) are the most revealing signs that SARS-CoV-2 evolved by natural evolution.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species (bats) are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published (\n\nAnother claim in Chinese social media points to a Nature Medicine paper published in 2015 [7] , which reports the construction of a chimeric CoV with a bat CoV S gene (SHC014) in the backbone of a SARS CoV that has adapted to infect mice (MA15) and is capable of infecting human cells [8] .", "There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published (\n\nAnother claim in Chinese social media points to a Nature Medicine paper published in 2015 [7] , which reports the construction of a chimeric CoV with a bat CoV S gene (SHC014) in the backbone of a SARS CoV that has adapted to infect mice (MA15) and is capable of infecting human cells [8] . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 (>5,000 nucleotides). The mouse-adapted SARS virus (MA15) [9] was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice.", "The mouse-adapted SARS virus (MA15) [9] was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice (hence M15), due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation.", "It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells [10, 11] . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans [6, 12] .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans [6, 12] . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry [8] . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV [13] , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts.", "Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV [13] , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV.", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice [7] . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function (GOF) studies under the US government-mandated pause policy ( The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature.", "Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function (GOF) studies under the US government-mandated pause policy ( The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups [5, 14] , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv (a manuscript sharing site prior to any peer review), claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random [15] .", "In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random [15] . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host.", "It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments.", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author(s). Susan R. Weiss" ]

[ "nan\n\nText: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 (COVID-19) ( com/novel-coronavirus-covid-19-portal/). According to what has been reported [1] [2] [3] , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome (SARS) caused by SARS-CoV.", "According to what has been reported [1] [2] [3] , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome (SARS) caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity [4, 5] . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin.", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV (RaTG13) was recently reported, which shared ∼96% homology with the SARS-CoV-2 [4] . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide (nt) variations (SNVs) identified across the genome [6] .", "However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide (nt) variations (SNVs) identified across the genome [6] . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV [4] , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species (bats) are the most revealing signs that SARS-CoV-2 evolved by natural evolution.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species (bats) are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published (\n\nAnother claim in Chinese social media points to a Nature Medicine paper published in 2015 [7] , which reports the construction of a chimeric CoV with a bat CoV S gene (SHC014) in the backbone of a SARS CoV that has adapted to infect mice (MA15) and is capable of infecting human cells [8] .", "There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published (\n\nAnother claim in Chinese social media points to a Nature Medicine paper published in 2015 [7] , which reports the construction of a chimeric CoV with a bat CoV S gene (SHC014) in the backbone of a SARS CoV that has adapted to infect mice (MA15) and is capable of infecting human cells [8] . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 (>5,000 nucleotides). The mouse-adapted SARS virus (MA15) [9] was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice.", "The mouse-adapted SARS virus (MA15) [9] was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice (hence M15), due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation.", "It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells [10, 11] . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans [6, 12] .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans [6, 12] . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry [8] . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV [13] , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts.", "Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV [13] , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV.", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice [7] . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function (GOF) studies under the US government-mandated pause policy ( The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature.", "Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function (GOF) studies under the US government-mandated pause policy ( The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups [5, 14] , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv (a manuscript sharing site prior to any peer review), claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random [15] .", "In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random [15] . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host.", "It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments.", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author(s). Susan R. Weiss" ]

[ "nan\n\nText: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 (COVID-19) ( com/novel-coronavirus-covid-19-portal/). According to what has been reported [1] [2] [3] , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome (SARS) caused by SARS-CoV.", "According to what has been reported [1] [2] [3] , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome (SARS) caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity [4, 5] . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin.", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV (RaTG13) was recently reported, which shared ∼96% homology with the SARS-CoV-2 [4] . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide (nt) variations (SNVs) identified across the genome [6] .", "However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide (nt) variations (SNVs) identified across the genome [6] . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV [4] , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species (bats) are the most revealing signs that SARS-CoV-2 evolved by natural evolution.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species (bats) are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published (\n\nAnother claim in Chinese social media points to a Nature Medicine paper published in 2015 [7] , which reports the construction of a chimeric CoV with a bat CoV S gene (SHC014) in the backbone of a SARS CoV that has adapted to infect mice (MA15) and is capable of infecting human cells [8] .", "There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published (\n\nAnother claim in Chinese social media points to a Nature Medicine paper published in 2015 [7] , which reports the construction of a chimeric CoV with a bat CoV S gene (SHC014) in the backbone of a SARS CoV that has adapted to infect mice (MA15) and is capable of infecting human cells [8] . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 (>5,000 nucleotides). The mouse-adapted SARS virus (MA15) [9] was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice.", "The mouse-adapted SARS virus (MA15) [9] was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice (hence M15), due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation.", "It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells [10, 11] . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans [6, 12] .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans [6, 12] . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry [8] . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV [13] , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts.", "Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV [13] , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV.", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice [7] . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function (GOF) studies under the US government-mandated pause policy ( The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature.", "Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function (GOF) studies under the US government-mandated pause policy ( The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups [5, 14] , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv (a manuscript sharing site prior to any peer review), claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random [15] .", "In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random [15] . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host.", "It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments.", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author(s). Susan R. Weiss" ]

[ "nan\n\nText: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 (COVID-19) ( com/novel-coronavirus-covid-19-portal/). According to what has been reported [1] [2] [3] , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome (SARS) caused by SARS-CoV.", "According to what has been reported [1] [2] [3] , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome (SARS) caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity [4, 5] . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin.", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV (RaTG13) was recently reported, which shared ∼96% homology with the SARS-CoV-2 [4] . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide (nt) variations (SNVs) identified across the genome [6] .", "However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide (nt) variations (SNVs) identified across the genome [6] . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV [4] , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species (bats) are the most revealing signs that SARS-CoV-2 evolved by natural evolution.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species (bats) are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published (\n\nAnother claim in Chinese social media points to a Nature Medicine paper published in 2015 [7] , which reports the construction of a chimeric CoV with a bat CoV S gene (SHC014) in the backbone of a SARS CoV that has adapted to infect mice (MA15) and is capable of infecting human cells [8] .", "There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published (\n\nAnother claim in Chinese social media points to a Nature Medicine paper published in 2015 [7] , which reports the construction of a chimeric CoV with a bat CoV S gene (SHC014) in the backbone of a SARS CoV that has adapted to infect mice (MA15) and is capable of infecting human cells [8] . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 (>5,000 nucleotides). The mouse-adapted SARS virus (MA15) [9] was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice.", "The mouse-adapted SARS virus (MA15) [9] was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice (hence M15), due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation.", "It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells [10, 11] . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans [6, 12] .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans [6, 12] . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry [8] . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV [13] , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts.", "Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV [13] , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV.", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice [7] . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function (GOF) studies under the US government-mandated pause policy ( The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature.", "Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function (GOF) studies under the US government-mandated pause policy ( The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups [5, 14] , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv (a manuscript sharing site prior to any peer review), claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random [15] .", "In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random [15] . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host.", "It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments.", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author(s). Susan R. Weiss" ]