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[ "Bats have been identified as a natural reservoir of a variety of coronaviruses (CoVs). Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance.", "Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV (BtCoV/Rh/YN2012) in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene.", "Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus.", "One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells.", "We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission.", "Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb [1] . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] .", "These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] .", "Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] . Six human CoVs (HCoVs) are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and\n\nAll sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201).", "HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and\n\nAll sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201). The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] .", "Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit (Roche Diagnostics GmbH, Mannheim, Germany) as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] .", "RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b (Cytob) and/or NADH dehydrogenase subunit 1 (ND1) gene using DNA extracted from the feces or swabs [19, 20] . The gene sequences were assembled excluding the primer sequences.", "The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request).", "Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request). Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends (SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA), respectively. PCR products were gel-purified and subjected directly to sequencing.", "PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) for sequencing. At least five independent clones were sequenced to obtain a consensus sequence.", "At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing (NGS) data were filtered and mapped to the reference sequence of BatCoV HKU10 (GenBank accession number NC_018871) using Geneious 7.1.8 [21] . Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA).", "Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA). Putative open reading frames (ORFs) were predicted using NCBI's ORF finder ( orffinder/) with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] .", "The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package [23] .", "Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package [23] . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE [24] . The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] .", "The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] . Potential recombination events as suggested by strong p-values (<10 -20 ) were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 [26] . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform ( with default parameters [27] .", "The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform ( with default parameters [27] . The protein homology detection was analyzed using HHpred ( with default parameters [28] . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] .", "A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing.", "Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells (Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT) generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS.", "Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection (ATCC, ). The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] .", "The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] . The influenza virus NS1 plasmid was generated in our lab [31] . The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences.", "The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies).", "The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies). Expression of these accessory genes were analyzed by Western blotting using an mAb (Roche Diagnostics GmbH, Mannheim, Germany) against the HA tag. The virus isolation was performed as previously described [12] .", "The virus isolation was performed as previously described [12] . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA).", "After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA). Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] .", "Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] . Apoptosis was analyzed as previously described [18] . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection.", "Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit (YEASEN, Shanghai, China) following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis.", "Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids (pRL-TK and pIFN-βIFN-or pNF-κB-Luc) [30] , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 [32] , SARS-CoV ORF7a [33] , or HBoV VP2 [34] . At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively.", "At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates.", "Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] .", "The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China.", "The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species ( Figure 1A ). CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ).", "CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ). Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity).", "Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species.", "These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species.", "These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively.", "It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained.", "Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD).", "Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD). The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ).", "The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ). Exceptions including three additional ORFs (ORF3b, ORF4a and ORF4b) were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b.", "All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16).", "It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16). The 3'-end of the cleavage sites recognized by 3C-like proteinase (Nsp4-Nsp10, Nsp12-Nsp16) and papain-like proteinase (Nsp1-Nsp3) were confirmed. The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ).", "The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ). The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs ( Table 2 ), suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013.", "The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes (83% genome identity) correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity).", "The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity). We then examined the individual genes ( Table 2) . All of the genes showed low aa sequence identity to known CoVs.", "All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab (>83.7% aa identity). Notably, the spike proteins are highly divergent among these strains.", "Notably, the spike proteins are highly divergent among these strains. Other structure proteins (E, M, and N) are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a.", "Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes (<65% aa identity). The putative accessory genes were also BLASTed against GenBank records.", "The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a (52.0-55.5% aa identity with BatCoV HKU10 ORF3) and ORF9 (28.1-32.0% aa identity with SARSr-CoV ORF7a). We analyzed the protein homology with HHpred software.", "We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues (possibility: 100%, E value <10 −48 ). We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis.", "No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ).", "The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs.", "Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described.", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS.", "The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b.", "In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios (Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site) were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection.", "The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 (0.727, 0.623, and 0.843, respectively) were significantly higher than those of other ORFs (Table 4 ). For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B \n\nAs SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a.", "For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B \n\nAs SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ).", "Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ). ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 ( Figure S2 ). The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction.", "The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids (pIFNβ-Luc and pRL-TK) and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection.", "All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression.", "Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ).", "Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot ( Figure S1 ). was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression.", "Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ).", "Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot (Figure S1 ). Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase.", "The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng).", "293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase.", "Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change).", "Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell.", "NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay.", "293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB.", "(B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α.", "After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently.", "The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test).", "Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB.", "In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids (pNF-κB-Luc and pRL-TK), as well as accessory protein-expressing plasmids, or controls (empty vector, NS1, SARS-CoV Tor2-ORF7a). The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection.", "The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 ( Figure 5B ). Expressions of ORF3as were confirmed with Western blot ( Figure S1 ).", "Expressions of ORF3as were confirmed with Western blot ( Figure S1 ). Other accessory proteins did not modulate NF-κB production ( Figure S4 ). To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies.", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully ( Figure S5 ). A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) .", "A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences.", "The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard [42] . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes.", "These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations.", "Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats.", "Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there.", "Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b (only in RsYN1), and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV.", "Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection [43] . In most CoVs, accessory genes are dispensable for virus replication.", "In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut [44] [45] [46] . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] .", "Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing [48] , inhibition of cellular protein synthesis [49] , cell-cycle blockage [50] , and apoptosis induction [51, 52] . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a.", "In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer.", "We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway.", "These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals.", "Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface.", "The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans.", "Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins.", "Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon.", "Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus.", "Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]

[ "Bats have been identified as a natural reservoir of a variety of coronaviruses (CoVs). Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance.", "Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV (BtCoV/Rh/YN2012) in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene.", "Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus.", "One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells.", "We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission.", "Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb [1] . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] .", "These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] .", "Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] . Six human CoVs (HCoVs) are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and\n\nAll sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201).", "HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and\n\nAll sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201). The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] .", "Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit (Roche Diagnostics GmbH, Mannheim, Germany) as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] .", "RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b (Cytob) and/or NADH dehydrogenase subunit 1 (ND1) gene using DNA extracted from the feces or swabs [19, 20] . The gene sequences were assembled excluding the primer sequences.", "The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request).", "Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request). Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends (SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA), respectively. PCR products were gel-purified and subjected directly to sequencing.", "PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) for sequencing. At least five independent clones were sequenced to obtain a consensus sequence.", "At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing (NGS) data were filtered and mapped to the reference sequence of BatCoV HKU10 (GenBank accession number NC_018871) using Geneious 7.1.8 [21] . Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA).", "Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA). Putative open reading frames (ORFs) were predicted using NCBI's ORF finder ( orffinder/) with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] .", "The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package [23] .", "Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package [23] . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE [24] . The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] .", "The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] . Potential recombination events as suggested by strong p-values (<10 -20 ) were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 [26] . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform ( with default parameters [27] .", "The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform ( with default parameters [27] . The protein homology detection was analyzed using HHpred ( with default parameters [28] . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] .", "A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing.", "Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells (Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT) generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS.", "Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection (ATCC, ). The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] .", "The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] . The influenza virus NS1 plasmid was generated in our lab [31] . The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences.", "The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies).", "The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies). Expression of these accessory genes were analyzed by Western blotting using an mAb (Roche Diagnostics GmbH, Mannheim, Germany) against the HA tag. The virus isolation was performed as previously described [12] .", "The virus isolation was performed as previously described [12] . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA).", "After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA). Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] .", "Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] . Apoptosis was analyzed as previously described [18] . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection.", "Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit (YEASEN, Shanghai, China) following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis.", "Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids (pRL-TK and pIFN-βIFN-or pNF-κB-Luc) [30] , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 [32] , SARS-CoV ORF7a [33] , or HBoV VP2 [34] . At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively.", "At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates.", "Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] .", "The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China.", "The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species ( Figure 1A ). CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ).", "CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ). Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity).", "Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species.", "These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species.", "These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively.", "It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained.", "Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD).", "Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD). The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ).", "The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ). Exceptions including three additional ORFs (ORF3b, ORF4a and ORF4b) were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b.", "All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16).", "It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16). The 3'-end of the cleavage sites recognized by 3C-like proteinase (Nsp4-Nsp10, Nsp12-Nsp16) and papain-like proteinase (Nsp1-Nsp3) were confirmed. The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ).", "The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ). The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs ( Table 2 ), suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013.", "The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes (83% genome identity) correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity).", "The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity). We then examined the individual genes ( Table 2) . All of the genes showed low aa sequence identity to known CoVs.", "All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab (>83.7% aa identity). Notably, the spike proteins are highly divergent among these strains.", "Notably, the spike proteins are highly divergent among these strains. Other structure proteins (E, M, and N) are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a.", "Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes (<65% aa identity). The putative accessory genes were also BLASTed against GenBank records.", "The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a (52.0-55.5% aa identity with BatCoV HKU10 ORF3) and ORF9 (28.1-32.0% aa identity with SARSr-CoV ORF7a). We analyzed the protein homology with HHpred software.", "We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues (possibility: 100%, E value <10 −48 ). We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis.", "No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ).", "The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs.", "Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described.", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS.", "The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b.", "In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios (Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site) were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection.", "The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 (0.727, 0.623, and 0.843, respectively) were significantly higher than those of other ORFs (Table 4 ). For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B \n\nAs SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a.", "For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B \n\nAs SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ).", "Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ). ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 ( Figure S2 ). The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction.", "The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids (pIFNβ-Luc and pRL-TK) and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection.", "All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression.", "Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ).", "Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot ( Figure S1 ). was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression.", "Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ).", "Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot (Figure S1 ). Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase.", "The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng).", "293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase.", "Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change).", "Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell.", "NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay.", "293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB.", "(B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α.", "After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently.", "The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test).", "Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB.", "In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids (pNF-κB-Luc and pRL-TK), as well as accessory protein-expressing plasmids, or controls (empty vector, NS1, SARS-CoV Tor2-ORF7a). The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection.", "The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 ( Figure 5B ). Expressions of ORF3as were confirmed with Western blot ( Figure S1 ).", "Expressions of ORF3as were confirmed with Western blot ( Figure S1 ). Other accessory proteins did not modulate NF-κB production ( Figure S4 ). To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies.", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully ( Figure S5 ). A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) .", "A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences.", "The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard [42] . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes.", "These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations.", "Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats.", "Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there.", "Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b (only in RsYN1), and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV.", "Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection [43] . In most CoVs, accessory genes are dispensable for virus replication.", "In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut [44] [45] [46] . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] .", "Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing [48] , inhibition of cellular protein synthesis [49] , cell-cycle blockage [50] , and apoptosis induction [51, 52] . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a.", "In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer.", "We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway.", "These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals.", "Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface.", "The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans.", "Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins.", "Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon.", "Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus.", "Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]

[ "Bats have been identified as a natural reservoir of a variety of coronaviruses (CoVs). Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance.", "Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV (BtCoV/Rh/YN2012) in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene.", "Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus.", "One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells.", "We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission.", "Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb [1] . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] .", "These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] .", "Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] . Six human CoVs (HCoVs) are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and\n\nAll sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201).", "HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and\n\nAll sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201). The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] .", "Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit (Roche Diagnostics GmbH, Mannheim, Germany) as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] .", "RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b (Cytob) and/or NADH dehydrogenase subunit 1 (ND1) gene using DNA extracted from the feces or swabs [19, 20] . The gene sequences were assembled excluding the primer sequences.", "The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request).", "Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request). Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends (SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA), respectively. PCR products were gel-purified and subjected directly to sequencing.", "PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) for sequencing. At least five independent clones were sequenced to obtain a consensus sequence.", "At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing (NGS) data were filtered and mapped to the reference sequence of BatCoV HKU10 (GenBank accession number NC_018871) using Geneious 7.1.8 [21] . Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA).", "Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA). Putative open reading frames (ORFs) were predicted using NCBI's ORF finder ( orffinder/) with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] .", "The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package [23] .", "Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package [23] . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE [24] . The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] .", "The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] . Potential recombination events as suggested by strong p-values (<10 -20 ) were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 [26] . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform ( with default parameters [27] .", "The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform ( with default parameters [27] . The protein homology detection was analyzed using HHpred ( with default parameters [28] . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] .", "A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing.", "Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells (Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT) generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS.", "Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection (ATCC, ). The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] .", "The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] . The influenza virus NS1 plasmid was generated in our lab [31] . The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences.", "The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies).", "The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies). Expression of these accessory genes were analyzed by Western blotting using an mAb (Roche Diagnostics GmbH, Mannheim, Germany) against the HA tag. The virus isolation was performed as previously described [12] .", "The virus isolation was performed as previously described [12] . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA).", "After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA). Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] .", "Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] . Apoptosis was analyzed as previously described [18] . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection.", "Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit (YEASEN, Shanghai, China) following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis.", "Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids (pRL-TK and pIFN-βIFN-or pNF-κB-Luc) [30] , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 [32] , SARS-CoV ORF7a [33] , or HBoV VP2 [34] . At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively.", "At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates.", "Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] .", "The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China.", "The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species ( Figure 1A ). CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ).", "CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ). Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity).", "Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species.", "These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species.", "These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively.", "It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained.", "Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD).", "Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD). The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ).", "The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ). Exceptions including three additional ORFs (ORF3b, ORF4a and ORF4b) were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b.", "All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16).", "It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16). The 3'-end of the cleavage sites recognized by 3C-like proteinase (Nsp4-Nsp10, Nsp12-Nsp16) and papain-like proteinase (Nsp1-Nsp3) were confirmed. The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ).", "The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ). The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs ( Table 2 ), suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013.", "The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes (83% genome identity) correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity).", "The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity). We then examined the individual genes ( Table 2) . All of the genes showed low aa sequence identity to known CoVs.", "All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab (>83.7% aa identity). Notably, the spike proteins are highly divergent among these strains.", "Notably, the spike proteins are highly divergent among these strains. Other structure proteins (E, M, and N) are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a.", "Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes (<65% aa identity). The putative accessory genes were also BLASTed against GenBank records.", "The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a (52.0-55.5% aa identity with BatCoV HKU10 ORF3) and ORF9 (28.1-32.0% aa identity with SARSr-CoV ORF7a). We analyzed the protein homology with HHpred software.", "We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues (possibility: 100%, E value <10 −48 ). We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis.", "No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ).", "The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs.", "Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described.", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS.", "The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b.", "In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios (Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site) were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection.", "The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 (0.727, 0.623, and 0.843, respectively) were significantly higher than those of other ORFs (Table 4 ). For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B \n\nAs SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a.", "For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B \n\nAs SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ).", "Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ). ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 ( Figure S2 ). The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction.", "The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids (pIFNβ-Luc and pRL-TK) and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection.", "All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression.", "Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ).", "Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot ( Figure S1 ). was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression.", "Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ).", "Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot (Figure S1 ). Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase.", "The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng).", "293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase.", "Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change).", "Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell.", "NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay.", "293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB.", "(B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α.", "After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently.", "The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test).", "Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB.", "In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids (pNF-κB-Luc and pRL-TK), as well as accessory protein-expressing plasmids, or controls (empty vector, NS1, SARS-CoV Tor2-ORF7a). The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection.", "The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 ( Figure 5B ). Expressions of ORF3as were confirmed with Western blot ( Figure S1 ).", "Expressions of ORF3as were confirmed with Western blot ( Figure S1 ). Other accessory proteins did not modulate NF-κB production ( Figure S4 ). To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies.", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully ( Figure S5 ). A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) .", "A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences.", "The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard [42] . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes.", "These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations.", "Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats.", "Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there.", "Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b (only in RsYN1), and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV.", "Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection [43] . In most CoVs, accessory genes are dispensable for virus replication.", "In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut [44] [45] [46] . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] .", "Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing [48] , inhibition of cellular protein synthesis [49] , cell-cycle blockage [50] , and apoptosis induction [51, 52] . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a.", "In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer.", "We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway.", "These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals.", "Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface.", "The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans.", "Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins.", "Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon.", "Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus.", "Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]

[ "Bats have been identified as a natural reservoir of a variety of coronaviruses (CoVs). Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance.", "Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV (BtCoV/Rh/YN2012) in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene.", "Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus.", "One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells.", "We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission.", "Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb [1] . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] .", "These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] .", "Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] . Six human CoVs (HCoVs) are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and\n\nAll sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201).", "HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and\n\nAll sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201). The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] .", "Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit (Roche Diagnostics GmbH, Mannheim, Germany) as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] .", "RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b (Cytob) and/or NADH dehydrogenase subunit 1 (ND1) gene using DNA extracted from the feces or swabs [19, 20] . The gene sequences were assembled excluding the primer sequences.", "The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request).", "Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request). Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends (SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA), respectively. PCR products were gel-purified and subjected directly to sequencing.", "PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) for sequencing. At least five independent clones were sequenced to obtain a consensus sequence.", "At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing (NGS) data were filtered and mapped to the reference sequence of BatCoV HKU10 (GenBank accession number NC_018871) using Geneious 7.1.8 [21] . Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA).", "Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA). Putative open reading frames (ORFs) were predicted using NCBI's ORF finder ( orffinder/) with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] .", "The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package [23] .", "Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package [23] . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE [24] . The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] .", "The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] . Potential recombination events as suggested by strong p-values (<10 -20 ) were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 [26] . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform ( with default parameters [27] .", "The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform ( with default parameters [27] . The protein homology detection was analyzed using HHpred ( with default parameters [28] . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] .", "A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing.", "Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells (Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT) generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS.", "Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection (ATCC, ). The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] .", "The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] . The influenza virus NS1 plasmid was generated in our lab [31] . The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences.", "The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies).", "The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies). Expression of these accessory genes were analyzed by Western blotting using an mAb (Roche Diagnostics GmbH, Mannheim, Germany) against the HA tag. The virus isolation was performed as previously described [12] .", "The virus isolation was performed as previously described [12] . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA).", "After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA). Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] .", "Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] . Apoptosis was analyzed as previously described [18] . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection.", "Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit (YEASEN, Shanghai, China) following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis.", "Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids (pRL-TK and pIFN-βIFN-or pNF-κB-Luc) [30] , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 [32] , SARS-CoV ORF7a [33] , or HBoV VP2 [34] . At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively.", "At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates.", "Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] .", "The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China.", "The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species ( Figure 1A ). CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ).", "CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ). Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity).", "Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species.", "These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species.", "These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively.", "It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained.", "Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD).", "Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD). The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ).", "The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ). Exceptions including three additional ORFs (ORF3b, ORF4a and ORF4b) were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b.", "All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16).", "It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16). The 3'-end of the cleavage sites recognized by 3C-like proteinase (Nsp4-Nsp10, Nsp12-Nsp16) and papain-like proteinase (Nsp1-Nsp3) were confirmed. The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ).", "The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ). The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs ( Table 2 ), suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013.", "The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes (83% genome identity) correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity).", "The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity). We then examined the individual genes ( Table 2) . All of the genes showed low aa sequence identity to known CoVs.", "All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab (>83.7% aa identity). Notably, the spike proteins are highly divergent among these strains.", "Notably, the spike proteins are highly divergent among these strains. Other structure proteins (E, M, and N) are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a.", "Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes (<65% aa identity). The putative accessory genes were also BLASTed against GenBank records.", "The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a (52.0-55.5% aa identity with BatCoV HKU10 ORF3) and ORF9 (28.1-32.0% aa identity with SARSr-CoV ORF7a). We analyzed the protein homology with HHpred software.", "We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues (possibility: 100%, E value <10 −48 ). We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis.", "No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ).", "The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs.", "Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described.", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS.", "The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b.", "In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios (Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site) were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection.", "The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 (0.727, 0.623, and 0.843, respectively) were significantly higher than those of other ORFs (Table 4 ). For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B \n\nAs SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a.", "For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B \n\nAs SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ).", "Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ). ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 ( Figure S2 ). The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction.", "The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids (pIFNβ-Luc and pRL-TK) and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection.", "All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression.", "Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ).", "Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot ( Figure S1 ). was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression.", "Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ).", "Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot (Figure S1 ). Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase.", "The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng).", "293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase.", "Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change).", "Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell.", "NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay.", "293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB.", "(B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α.", "After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently.", "The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test).", "Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB.", "In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids (pNF-κB-Luc and pRL-TK), as well as accessory protein-expressing plasmids, or controls (empty vector, NS1, SARS-CoV Tor2-ORF7a). The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection.", "The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 ( Figure 5B ). Expressions of ORF3as were confirmed with Western blot ( Figure S1 ).", "Expressions of ORF3as were confirmed with Western blot ( Figure S1 ). Other accessory proteins did not modulate NF-κB production ( Figure S4 ). To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies.", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully ( Figure S5 ). A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) .", "A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences.", "The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard [42] . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes.", "These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations.", "Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats.", "Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there.", "Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b (only in RsYN1), and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV.", "Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection [43] . In most CoVs, accessory genes are dispensable for virus replication.", "In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut [44] [45] [46] . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] .", "Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing [48] , inhibition of cellular protein synthesis [49] , cell-cycle blockage [50] , and apoptosis induction [51, 52] . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a.", "In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer.", "We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway.", "These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals.", "Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface.", "The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans.", "Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins.", "Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon.", "Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus.", "Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]

[ "Bats have been identified as a natural reservoir of a variety of coronaviruses (CoVs). Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance.", "Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV (BtCoV/Rh/YN2012) in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene.", "Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus.", "One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells.", "We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission.", "Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb [1] . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] .", "These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] .", "Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] . Six human CoVs (HCoVs) are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and\n\nAll sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201).", "HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and\n\nAll sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201). The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] .", "Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit (Roche Diagnostics GmbH, Mannheim, Germany) as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] .", "RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b (Cytob) and/or NADH dehydrogenase subunit 1 (ND1) gene using DNA extracted from the feces or swabs [19, 20] . The gene sequences were assembled excluding the primer sequences.", "The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request).", "Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request). Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends (SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA), respectively. PCR products were gel-purified and subjected directly to sequencing.", "PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) for sequencing. At least five independent clones were sequenced to obtain a consensus sequence.", "At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing (NGS) data were filtered and mapped to the reference sequence of BatCoV HKU10 (GenBank accession number NC_018871) using Geneious 7.1.8 [21] . Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA).", "Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA). Putative open reading frames (ORFs) were predicted using NCBI's ORF finder ( orffinder/) with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] .", "The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package [23] .", "Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package [23] . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE [24] . The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] .", "The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] . Potential recombination events as suggested by strong p-values (<10 -20 ) were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 [26] . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform ( with default parameters [27] .", "The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform ( with default parameters [27] . The protein homology detection was analyzed using HHpred ( with default parameters [28] . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] .", "A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing.", "Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells (Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT) generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS.", "Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection (ATCC, ). The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] .", "The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] . The influenza virus NS1 plasmid was generated in our lab [31] . The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences.", "The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies).", "The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies). Expression of these accessory genes were analyzed by Western blotting using an mAb (Roche Diagnostics GmbH, Mannheim, Germany) against the HA tag. The virus isolation was performed as previously described [12] .", "The virus isolation was performed as previously described [12] . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA).", "After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA). Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] .", "Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] . Apoptosis was analyzed as previously described [18] . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection.", "Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit (YEASEN, Shanghai, China) following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis.", "Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids (pRL-TK and pIFN-βIFN-or pNF-κB-Luc) [30] , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 [32] , SARS-CoV ORF7a [33] , or HBoV VP2 [34] . At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively.", "At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates.", "Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] .", "The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China.", "The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species ( Figure 1A ). CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ).", "CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ). Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity).", "Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species.", "These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species.", "These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively.", "It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained.", "Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD).", "Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD). The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ).", "The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ). Exceptions including three additional ORFs (ORF3b, ORF4a and ORF4b) were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b.", "All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16).", "It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16). The 3'-end of the cleavage sites recognized by 3C-like proteinase (Nsp4-Nsp10, Nsp12-Nsp16) and papain-like proteinase (Nsp1-Nsp3) were confirmed. The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ).", "The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ). The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs ( Table 2 ), suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013.", "The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes (83% genome identity) correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity).", "The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity). We then examined the individual genes ( Table 2) . All of the genes showed low aa sequence identity to known CoVs.", "All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab (>83.7% aa identity). Notably, the spike proteins are highly divergent among these strains.", "Notably, the spike proteins are highly divergent among these strains. Other structure proteins (E, M, and N) are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a.", "Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes (<65% aa identity). The putative accessory genes were also BLASTed against GenBank records.", "The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a (52.0-55.5% aa identity with BatCoV HKU10 ORF3) and ORF9 (28.1-32.0% aa identity with SARSr-CoV ORF7a). We analyzed the protein homology with HHpred software.", "We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues (possibility: 100%, E value <10 −48 ). We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis.", "No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ).", "The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs.", "Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described.", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS.", "The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b.", "In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios (Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site) were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection.", "The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 (0.727, 0.623, and 0.843, respectively) were significantly higher than those of other ORFs (Table 4 ). For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B \n\nAs SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a.", "For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B \n\nAs SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ).", "Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ). ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 ( Figure S2 ). The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction.", "The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids (pIFNβ-Luc and pRL-TK) and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection.", "All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression.", "Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ).", "Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot ( Figure S1 ). was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression.", "Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ).", "Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot (Figure S1 ). Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase.", "The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng).", "293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase.", "Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change).", "Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell.", "NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay.", "293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB.", "(B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α.", "After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently.", "The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test).", "Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB.", "In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids (pNF-κB-Luc and pRL-TK), as well as accessory protein-expressing plasmids, or controls (empty vector, NS1, SARS-CoV Tor2-ORF7a). The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection.", "The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 ( Figure 5B ). Expressions of ORF3as were confirmed with Western blot ( Figure S1 ).", "Expressions of ORF3as were confirmed with Western blot ( Figure S1 ). Other accessory proteins did not modulate NF-κB production ( Figure S4 ). To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies.", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully ( Figure S5 ). A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) .", "A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences.", "The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard [42] . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes.", "These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations.", "Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats.", "Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there.", "Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b (only in RsYN1), and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV.", "Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection [43] . In most CoVs, accessory genes are dispensable for virus replication.", "In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut [44] [45] [46] . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] .", "Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing [48] , inhibition of cellular protein synthesis [49] , cell-cycle blockage [50] , and apoptosis induction [51, 52] . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a.", "In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer.", "We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway.", "These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals.", "Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface.", "The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans.", "Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins.", "Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon.", "Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus.", "Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]

[ "Bats have been identified as a natural reservoir of a variety of coronaviruses (CoVs). Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance.", "Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV (BtCoV/Rh/YN2012) in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene.", "Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus.", "One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells.", "We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission.", "Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb [1] . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] .", "These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] .", "Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] . Six human CoVs (HCoVs) are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and\n\nAll sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201).", "HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and\n\nAll sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201). The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] .", "Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit (Roche Diagnostics GmbH, Mannheim, Germany) as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] .", "RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b (Cytob) and/or NADH dehydrogenase subunit 1 (ND1) gene using DNA extracted from the feces or swabs [19, 20] . The gene sequences were assembled excluding the primer sequences.", "The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request).", "Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request). Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends (SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA), respectively. PCR products were gel-purified and subjected directly to sequencing.", "PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) for sequencing. At least five independent clones were sequenced to obtain a consensus sequence.", "At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing (NGS) data were filtered and mapped to the reference sequence of BatCoV HKU10 (GenBank accession number NC_018871) using Geneious 7.1.8 [21] . Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA).", "Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA). Putative open reading frames (ORFs) were predicted using NCBI's ORF finder ( orffinder/) with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] .", "The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package [23] .", "Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package [23] . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE [24] . The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] .", "The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] . Potential recombination events as suggested by strong p-values (<10 -20 ) were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 [26] . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform ( with default parameters [27] .", "The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform ( with default parameters [27] . The protein homology detection was analyzed using HHpred ( with default parameters [28] . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] .", "A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing.", "Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells (Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT) generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS.", "Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection (ATCC, ). The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] .", "The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] . The influenza virus NS1 plasmid was generated in our lab [31] . The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences.", "The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies).", "The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies). Expression of these accessory genes were analyzed by Western blotting using an mAb (Roche Diagnostics GmbH, Mannheim, Germany) against the HA tag. The virus isolation was performed as previously described [12] .", "The virus isolation was performed as previously described [12] . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA).", "After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA). Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] .", "Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] . Apoptosis was analyzed as previously described [18] . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection.", "Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit (YEASEN, Shanghai, China) following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis.", "Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids (pRL-TK and pIFN-βIFN-or pNF-κB-Luc) [30] , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 [32] , SARS-CoV ORF7a [33] , or HBoV VP2 [34] . At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively.", "At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates.", "Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] .", "The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China.", "The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species ( Figure 1A ). CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ).", "CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ). Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity).", "Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species.", "These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species.", "These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively.", "It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained.", "Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD).", "Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD). The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ).", "The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ). Exceptions including three additional ORFs (ORF3b, ORF4a and ORF4b) were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b.", "All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16).", "It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16). The 3'-end of the cleavage sites recognized by 3C-like proteinase (Nsp4-Nsp10, Nsp12-Nsp16) and papain-like proteinase (Nsp1-Nsp3) were confirmed. The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ).", "The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ). The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs ( Table 2 ), suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013.", "The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes (83% genome identity) correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity).", "The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity). We then examined the individual genes ( Table 2) . All of the genes showed low aa sequence identity to known CoVs.", "All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab (>83.7% aa identity). Notably, the spike proteins are highly divergent among these strains.", "Notably, the spike proteins are highly divergent among these strains. Other structure proteins (E, M, and N) are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a.", "Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes (<65% aa identity). The putative accessory genes were also BLASTed against GenBank records.", "The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a (52.0-55.5% aa identity with BatCoV HKU10 ORF3) and ORF9 (28.1-32.0% aa identity with SARSr-CoV ORF7a). We analyzed the protein homology with HHpred software.", "We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues (possibility: 100%, E value <10 −48 ). We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis.", "No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ).", "The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs.", "Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described.", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS.", "The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b.", "In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios (Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site) were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection.", "The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 (0.727, 0.623, and 0.843, respectively) were significantly higher than those of other ORFs (Table 4 ). For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B \n\nAs SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a.", "For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B \n\nAs SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ).", "Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ). ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 ( Figure S2 ). The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction.", "The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids (pIFNβ-Luc and pRL-TK) and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection.", "All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression.", "Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ).", "Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot ( Figure S1 ). was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression.", "Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ).", "Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot (Figure S1 ). Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase.", "The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng).", "293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase.", "Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change).", "Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell.", "NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay.", "293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB.", "(B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α.", "After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently.", "The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test).", "Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB.", "In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids (pNF-κB-Luc and pRL-TK), as well as accessory protein-expressing plasmids, or controls (empty vector, NS1, SARS-CoV Tor2-ORF7a). The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection.", "The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 ( Figure 5B ). Expressions of ORF3as were confirmed with Western blot ( Figure S1 ).", "Expressions of ORF3as were confirmed with Western blot ( Figure S1 ). Other accessory proteins did not modulate NF-κB production ( Figure S4 ). To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies.", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully ( Figure S5 ). A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) .", "A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences.", "The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard [42] . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes.", "These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations.", "Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats.", "Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there.", "Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b (only in RsYN1), and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV.", "Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection [43] . In most CoVs, accessory genes are dispensable for virus replication.", "In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut [44] [45] [46] . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] .", "Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing [48] , inhibition of cellular protein synthesis [49] , cell-cycle blockage [50] , and apoptosis induction [51, 52] . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a.", "In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer.", "We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway.", "These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals.", "Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface.", "The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans.", "Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins.", "Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon.", "Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus.", "Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]

[ "Bats have been identified as a natural reservoir of a variety of coronaviruses (CoVs). Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance.", "Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV (BtCoV/Rh/YN2012) in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene.", "Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus.", "One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells.", "We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission.", "Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb [1] . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] .", "These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] .", "Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] . Six human CoVs (HCoVs) are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and\n\nAll sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201).", "HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and\n\nAll sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201). The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] .", "Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit (Roche Diagnostics GmbH, Mannheim, Germany) as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] .", "RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b (Cytob) and/or NADH dehydrogenase subunit 1 (ND1) gene using DNA extracted from the feces or swabs [19, 20] . The gene sequences were assembled excluding the primer sequences.", "The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request).", "Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request). Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends (SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA), respectively. PCR products were gel-purified and subjected directly to sequencing.", "PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) for sequencing. At least five independent clones were sequenced to obtain a consensus sequence.", "At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing (NGS) data were filtered and mapped to the reference sequence of BatCoV HKU10 (GenBank accession number NC_018871) using Geneious 7.1.8 [21] . Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA).", "Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA). Putative open reading frames (ORFs) were predicted using NCBI's ORF finder ( orffinder/) with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] .", "The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package [23] .", "Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package [23] . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE [24] . The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] .", "The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] . Potential recombination events as suggested by strong p-values (<10 -20 ) were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 [26] . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform ( with default parameters [27] .", "The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform ( with default parameters [27] . The protein homology detection was analyzed using HHpred ( with default parameters [28] . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] .", "A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing.", "Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells (Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT) generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS.", "Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection (ATCC, ). The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] .", "The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] . The influenza virus NS1 plasmid was generated in our lab [31] . The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences.", "The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies).", "The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies). Expression of these accessory genes were analyzed by Western blotting using an mAb (Roche Diagnostics GmbH, Mannheim, Germany) against the HA tag. The virus isolation was performed as previously described [12] .", "The virus isolation was performed as previously described [12] . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA).", "After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA). Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] .", "Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] . Apoptosis was analyzed as previously described [18] . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection.", "Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit (YEASEN, Shanghai, China) following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis.", "Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids (pRL-TK and pIFN-βIFN-or pNF-κB-Luc) [30] , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 [32] , SARS-CoV ORF7a [33] , or HBoV VP2 [34] . At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively.", "At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates.", "Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] .", "The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China.", "The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species ( Figure 1A ). CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ).", "CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ). Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity).", "Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species.", "These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species.", "These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively.", "It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained.", "Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD).", "Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD). The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ).", "The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ). Exceptions including three additional ORFs (ORF3b, ORF4a and ORF4b) were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b.", "All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16).", "It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16). The 3'-end of the cleavage sites recognized by 3C-like proteinase (Nsp4-Nsp10, Nsp12-Nsp16) and papain-like proteinase (Nsp1-Nsp3) were confirmed. The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ).", "The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ). The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs ( Table 2 ), suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013.", "The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes (83% genome identity) correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity).", "The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity). We then examined the individual genes ( Table 2) . All of the genes showed low aa sequence identity to known CoVs.", "All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab (>83.7% aa identity). Notably, the spike proteins are highly divergent among these strains.", "Notably, the spike proteins are highly divergent among these strains. Other structure proteins (E, M, and N) are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a.", "Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes (<65% aa identity). The putative accessory genes were also BLASTed against GenBank records.", "The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a (52.0-55.5% aa identity with BatCoV HKU10 ORF3) and ORF9 (28.1-32.0% aa identity with SARSr-CoV ORF7a). We analyzed the protein homology with HHpred software.", "We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues (possibility: 100%, E value <10 −48 ). We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis.", "No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ).", "The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs.", "Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described.", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS.", "The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b.", "In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios (Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site) were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection.", "The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 (0.727, 0.623, and 0.843, respectively) were significantly higher than those of other ORFs (Table 4 ). For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B \n\nAs SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a.", "For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B \n\nAs SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ).", "Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ). ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 ( Figure S2 ). The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction.", "The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids (pIFNβ-Luc and pRL-TK) and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection.", "All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression.", "Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ).", "Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot ( Figure S1 ). was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression.", "Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ).", "Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot (Figure S1 ). Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase.", "The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng).", "293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase.", "Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change).", "Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell.", "NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay.", "293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB.", "(B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α.", "After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently.", "The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test).", "Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB.", "In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids (pNF-κB-Luc and pRL-TK), as well as accessory protein-expressing plasmids, or controls (empty vector, NS1, SARS-CoV Tor2-ORF7a). The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection.", "The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 ( Figure 5B ). Expressions of ORF3as were confirmed with Western blot ( Figure S1 ).", "Expressions of ORF3as were confirmed with Western blot ( Figure S1 ). Other accessory proteins did not modulate NF-κB production ( Figure S4 ). To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies.", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully ( Figure S5 ). A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) .", "A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences.", "The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard [42] . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes.", "These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations.", "Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats.", "Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there.", "Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b (only in RsYN1), and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV.", "Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection [43] . In most CoVs, accessory genes are dispensable for virus replication.", "In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut [44] [45] [46] . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] .", "Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing [48] , inhibition of cellular protein synthesis [49] , cell-cycle blockage [50] , and apoptosis induction [51, 52] . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a.", "In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer.", "We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway.", "These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals.", "Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface.", "The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans.", "Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins.", "Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon.", "Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus.", "Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]

[ "Bats have been identified as a natural reservoir of a variety of coronaviruses (CoVs). Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance.", "Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV (BtCoV/Rh/YN2012) in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene.", "Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus.", "One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells.", "We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission.", "Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb [1] . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] .", "These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] .", "Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] . Six human CoVs (HCoVs) are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and\n\nAll sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201).", "HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and\n\nAll sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201). The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] .", "Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit (Roche Diagnostics GmbH, Mannheim, Germany) as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] .", "RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b (Cytob) and/or NADH dehydrogenase subunit 1 (ND1) gene using DNA extracted from the feces or swabs [19, 20] . The gene sequences were assembled excluding the primer sequences.", "The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request).", "Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request). Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends (SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA), respectively. PCR products were gel-purified and subjected directly to sequencing.", "PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) for sequencing. At least five independent clones were sequenced to obtain a consensus sequence.", "At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing (NGS) data were filtered and mapped to the reference sequence of BatCoV HKU10 (GenBank accession number NC_018871) using Geneious 7.1.8 [21] . Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA).", "Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA). Putative open reading frames (ORFs) were predicted using NCBI's ORF finder ( orffinder/) with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] .", "The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package [23] .", "Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package [23] . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE [24] . The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] .", "The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] . Potential recombination events as suggested by strong p-values (<10 -20 ) were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 [26] . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform ( with default parameters [27] .", "The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform ( with default parameters [27] . The protein homology detection was analyzed using HHpred ( with default parameters [28] . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] .", "A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing.", "Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells (Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT) generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS.", "Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection (ATCC, ). The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] .", "The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] . The influenza virus NS1 plasmid was generated in our lab [31] . The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences.", "The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies).", "The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies). Expression of these accessory genes were analyzed by Western blotting using an mAb (Roche Diagnostics GmbH, Mannheim, Germany) against the HA tag. The virus isolation was performed as previously described [12] .", "The virus isolation was performed as previously described [12] . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA).", "After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA). Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] .", "Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] . Apoptosis was analyzed as previously described [18] . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection.", "Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit (YEASEN, Shanghai, China) following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis.", "Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids (pRL-TK and pIFN-βIFN-or pNF-κB-Luc) [30] , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 [32] , SARS-CoV ORF7a [33] , or HBoV VP2 [34] . At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively.", "At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates.", "Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] .", "The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China.", "The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species ( Figure 1A ). CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ).", "CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ). Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity).", "Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species.", "These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species.", "These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively.", "It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained.", "Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD).", "Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD). The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ).", "The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ). Exceptions including three additional ORFs (ORF3b, ORF4a and ORF4b) were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b.", "All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16).", "It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16). The 3'-end of the cleavage sites recognized by 3C-like proteinase (Nsp4-Nsp10, Nsp12-Nsp16) and papain-like proteinase (Nsp1-Nsp3) were confirmed. The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ).", "The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ). The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs ( Table 2 ), suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013.", "The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes (83% genome identity) correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity).", "The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity). We then examined the individual genes ( Table 2) . All of the genes showed low aa sequence identity to known CoVs.", "All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab (>83.7% aa identity). Notably, the spike proteins are highly divergent among these strains.", "Notably, the spike proteins are highly divergent among these strains. Other structure proteins (E, M, and N) are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a.", "Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes (<65% aa identity). The putative accessory genes were also BLASTed against GenBank records.", "The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a (52.0-55.5% aa identity with BatCoV HKU10 ORF3) and ORF9 (28.1-32.0% aa identity with SARSr-CoV ORF7a). We analyzed the protein homology with HHpred software.", "We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues (possibility: 100%, E value <10 −48 ). We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis.", "No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ).", "The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs.", "Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described.", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS.", "The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b.", "In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios (Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site) were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection.", "The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 (0.727, 0.623, and 0.843, respectively) were significantly higher than those of other ORFs (Table 4 ). For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B \n\nAs SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a.", "For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B \n\nAs SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ).", "Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ). ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 ( Figure S2 ). The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction.", "The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids (pIFNβ-Luc and pRL-TK) and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection.", "All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression.", "Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ).", "Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot ( Figure S1 ). was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression.", "Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ).", "Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot (Figure S1 ). Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase.", "The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng).", "293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase.", "Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change).", "Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell.", "NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay.", "293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB.", "(B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α.", "After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently.", "The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test).", "Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB.", "In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids (pNF-κB-Luc and pRL-TK), as well as accessory protein-expressing plasmids, or controls (empty vector, NS1, SARS-CoV Tor2-ORF7a). The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection.", "The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 ( Figure 5B ). Expressions of ORF3as were confirmed with Western blot ( Figure S1 ).", "Expressions of ORF3as were confirmed with Western blot ( Figure S1 ). Other accessory proteins did not modulate NF-κB production ( Figure S4 ). To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies.", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully ( Figure S5 ). A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) .", "A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences.", "The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard [42] . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes.", "These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations.", "Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats.", "Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there.", "Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b (only in RsYN1), and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV.", "Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection [43] . In most CoVs, accessory genes are dispensable for virus replication.", "In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut [44] [45] [46] . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] .", "Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing [48] , inhibition of cellular protein synthesis [49] , cell-cycle blockage [50] , and apoptosis induction [51, 52] . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a.", "In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer.", "We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway.", "These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals.", "Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface.", "The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans.", "Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins.", "Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon.", "Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus.", "Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]

[ "Bats have been identified as a natural reservoir of a variety of coronaviruses (CoVs). Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance.", "Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV (BtCoV/Rh/YN2012) in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene.", "Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus.", "One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells.", "We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission.", "Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb [1] . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] .", "These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] .", "Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] . Six human CoVs (HCoVs) are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and\n\nAll sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201).", "HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and\n\nAll sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201). The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] .", "Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit (Roche Diagnostics GmbH, Mannheim, Germany) as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] .", "RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b (Cytob) and/or NADH dehydrogenase subunit 1 (ND1) gene using DNA extracted from the feces or swabs [19, 20] . The gene sequences were assembled excluding the primer sequences.", "The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request).", "Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request). Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends (SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA), respectively. PCR products were gel-purified and subjected directly to sequencing.", "PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) for sequencing. At least five independent clones were sequenced to obtain a consensus sequence.", "At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing (NGS) data were filtered and mapped to the reference sequence of BatCoV HKU10 (GenBank accession number NC_018871) using Geneious 7.1.8 [21] . Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA).", "Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA). Putative open reading frames (ORFs) were predicted using NCBI's ORF finder ( orffinder/) with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] .", "The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package [23] .", "Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package [23] . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE [24] . The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] .", "The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] . Potential recombination events as suggested by strong p-values (<10 -20 ) were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 [26] . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform ( with default parameters [27] .", "The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform ( with default parameters [27] . The protein homology detection was analyzed using HHpred ( with default parameters [28] . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] .", "A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing.", "Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells (Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT) generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS.", "Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection (ATCC, ). The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] .", "The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] . The influenza virus NS1 plasmid was generated in our lab [31] . The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences.", "The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies).", "The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies). Expression of these accessory genes were analyzed by Western blotting using an mAb (Roche Diagnostics GmbH, Mannheim, Germany) against the HA tag. The virus isolation was performed as previously described [12] .", "The virus isolation was performed as previously described [12] . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA).", "After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA). Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] .", "Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] . Apoptosis was analyzed as previously described [18] . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection.", "Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit (YEASEN, Shanghai, China) following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis.", "Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids (pRL-TK and pIFN-βIFN-or pNF-κB-Luc) [30] , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 [32] , SARS-CoV ORF7a [33] , or HBoV VP2 [34] . At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively.", "At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates.", "Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] .", "The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China.", "The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species ( Figure 1A ). CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ).", "CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ). Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity).", "Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species.", "These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species.", "These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively.", "It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained.", "Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD).", "Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD). The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ).", "The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ). Exceptions including three additional ORFs (ORF3b, ORF4a and ORF4b) were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b.", "All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16).", "It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16). The 3'-end of the cleavage sites recognized by 3C-like proteinase (Nsp4-Nsp10, Nsp12-Nsp16) and papain-like proteinase (Nsp1-Nsp3) were confirmed. The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ).", "The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ). The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs ( Table 2 ), suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013.", "The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes (83% genome identity) correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity).", "The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity). We then examined the individual genes ( Table 2) . All of the genes showed low aa sequence identity to known CoVs.", "All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab (>83.7% aa identity). Notably, the spike proteins are highly divergent among these strains.", "Notably, the spike proteins are highly divergent among these strains. Other structure proteins (E, M, and N) are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a.", "Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes (<65% aa identity). The putative accessory genes were also BLASTed against GenBank records.", "The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a (52.0-55.5% aa identity with BatCoV HKU10 ORF3) and ORF9 (28.1-32.0% aa identity with SARSr-CoV ORF7a). We analyzed the protein homology with HHpred software.", "We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues (possibility: 100%, E value <10 −48 ). We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis.", "No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ).", "The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs.", "Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described.", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS.", "The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b.", "In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios (Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site) were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection.", "The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 (0.727, 0.623, and 0.843, respectively) were significantly higher than those of other ORFs (Table 4 ). For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B \n\nAs SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a.", "For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B \n\nAs SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ).", "Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ). ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 ( Figure S2 ). The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction.", "The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids (pIFNβ-Luc and pRL-TK) and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection.", "All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression.", "Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ).", "Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot ( Figure S1 ). was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression.", "Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ).", "Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot (Figure S1 ). Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase.", "The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng).", "293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase.", "Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change).", "Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell.", "NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay.", "293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB.", "(B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α.", "After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently.", "The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test).", "Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB.", "In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids (pNF-κB-Luc and pRL-TK), as well as accessory protein-expressing plasmids, or controls (empty vector, NS1, SARS-CoV Tor2-ORF7a). The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection.", "The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 ( Figure 5B ). Expressions of ORF3as were confirmed with Western blot ( Figure S1 ).", "Expressions of ORF3as were confirmed with Western blot ( Figure S1 ). Other accessory proteins did not modulate NF-κB production ( Figure S4 ). To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies.", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully ( Figure S5 ). A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) .", "A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences.", "The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard [42] . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes.", "These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations.", "Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats.", "Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there.", "Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b (only in RsYN1), and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV.", "Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection [43] . In most CoVs, accessory genes are dispensable for virus replication.", "In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut [44] [45] [46] . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] .", "Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing [48] , inhibition of cellular protein synthesis [49] , cell-cycle blockage [50] , and apoptosis induction [51, 52] . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a.", "In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer.", "We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway.", "These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals.", "Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface.", "The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans.", "Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins.", "Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon.", "Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus.", "Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]

[ "Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective.", "Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells.", "RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay.", "The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Text: Feline Infectious Peritonitis Virus (FIPV) is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome.", "Text: Feline Infectious Peritonitis Virus (FIPV) is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome. FIPV is grouped as feline coronavirus (FCoV), under the family Coronaviridae. FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus (FECV), a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV [1] .", "FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus (FECV), a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV [1] . The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory.", "Two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory. Internal mutation theory stated that the development of FIP is due to the exposure of cat to variants of FCoV which have been mutated by gaining the ability to replicate within the macrophages [2] , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population [3] . Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces [4] .", "Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces [4] . About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease [4] . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV.", "The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV. Vaccination against FIPV with an attenuated, temperature-sensitive strain of type II FIPV induces low antibody titre in kittens that have not been exposed to FCoV. However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] .", "However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] . In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement [1] . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] .", "Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] . However, those treatments were ineffective. Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection.", "Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection. Triple Helix Forming Oligonucleotide (TFO) is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA [9] . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] .", "Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] . Hence, TFO has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes [11] [12] [13] [14] [15] [16] . The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication.", "The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication. serotype II strain WSU 79-1146 (ATCC no. VR-1777) was grown in CRFK cells. A serial 10-fold dilution of FIPV was prepared from the working stock.", "A serial 10-fold dilution of FIPV was prepared from the working stock. Confluent 96-well plate was inoculated with 100 L of each virus dilution/well. The plate was incubated in a humidified incubator at 37 ∘ C, 5% CO 2 . Cytopathic effects (CPE) development was observed.", "Cytopathic effects (CPE) development was observed. The results were recorded after 72 hours and the virus tissue culture infective dose 50 (TCID 50 ) was calculated using Reed and Muench's method [17] . Oligonucleotide RNA.", "Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides (TFOs) were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 (Accession no: AY994055) [18] . TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed ( Table 1 ).", "TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed ( Table 1 ). The specificity of the TFOs was identified using BLAST search in the NCBI database. The designed linear TFOs were synthesized by Dharmacon Research (USA), whereby the 5 and 3 ends of the linear TFOs were modified with phosphate (PO 4 ) group and hydroxide (OH) group, respectively.", "The designed linear TFOs were synthesized by Dharmacon Research (USA), whereby the 5 and 3 ends of the linear TFOs were modified with phosphate (PO 4 ) group and hydroxide (OH) group, respectively. These modifications were necessary for the circularization of linear TFO. The process of circularization, using the T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs Inc., England), was carried out according to the manufacturer's protocol.", "The process of circularization, using the T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs Inc., England), was carried out according to the manufacturer's protocol. After ligation, the circular TFO RNAs were recovered by ethanol precipitation and the purity of the circular TFO RNAs was measured using spectrophotometer. Denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification.", "Denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. Then, the gel was prerun at 20 to 40 V for 45 minutes.", "Then, the gel was prerun at 20 to 40 V for 45 minutes. Five L of TFO RNA mixed with 5 L of urea loading buffer was heated at 92 ∘ C for 2 minutes and immediately chilled on ice. It was run on the gel at 200 V for 45 minutes.", "It was run on the gel at 200 V for 45 minutes. Finally, the gel was stained with ethidium bromide (Sigma, USA) and viewed with a Bio-Rad Gel Doc XR system (CA, USA). (EMSA) .", "(EMSA) . The target regions of the FIPV genome were synthesized by Dharmacon Research (USA) ( Table 1) . Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours.", "Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours. The sample was run on 15% native polyacrylamide gel at 80 V, in cool condition. The stained gel was viewed by a Bio-Rad Gel Doc XR system.", "The stained gel was viewed by a Bio-Rad Gel Doc XR system. Regions. The binding strength was measured using a nano Isothermal Titration Calorimeter (ITC) (TA instruments, Newcastle, UK).", "The binding strength was measured using a nano Isothermal Titration Calorimeter (ITC) (TA instruments, Newcastle, UK). The RNA sample mixtures, consisting of circular TFOs (0.0002 mM), were incubated with their respective synthetic target regions (0.015 mM) using 1X binding buffer as the diluent. The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections.", "The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer. This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection.", "This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. One hundred nM of TFO RNAs was separately transfected into the CRFK cells using a HiPerFect Transfection Reagent (Qiagen, Germany), as per the manufacturer's protocol. The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours.", "The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C (100 L/well). Finally, the viral inoculum was replaced by fresh maintenance media (MEM containing 1% FBS and 1% pen/strep).", "Finally, the viral inoculum was replaced by fresh maintenance media (MEM containing 1% FBS and 1% pen/strep). Virus-infected and uninfected cells were maintained as positive and negative controls, respectively. The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction.", "The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction. Inhibition. Different concentrations of circular TFO1 RNA (25 nM, 50 nM, 100 nM, and 500 nM) were transfected into CRFK cells.", "Different concentrations of circular TFO1 RNA (25 nM, 50 nM, 100 nM, and 500 nM) were transfected into CRFK cells. The plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ C with 5% CO2. The cells were processed as described above.", "The cells were processed as described above. Madin-Darby Canine Kidney (MDCK) cell (ATCC no. CCL-34), at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. Transfection was performed the same as before.", "Transfection was performed the same as before. One hundred nM of circular TFO RNA was transfected into MDCK cells. Following 6 hours \n\nORF1a/1b and 530-541\n\nORF1a/1b and 7399-7411\n\nORF1a/1b and 14048-14061\n\n- * Highlighted in bold indicated the binding region. * * Unrelated circular TFO. [20, 21] , respectively.", "* * Unrelated circular TFO. [20, 21] , respectively. The reverse transcriptase quantitative real-time PCR (RT-qPCR) was performed using a Bio-Rad CFX96 real-time system (BioRad, USA).", "The reverse transcriptase quantitative real-time PCR (RT-qPCR) was performed using a Bio-Rad CFX96 real-time system (BioRad, USA). The reaction was amplified in a final volume of 25 L using a SensiMix SYBR No-ROX One-Step Kit (Bioline, UK), which consisted of 12.5 L 2X SensiMix SYBR No-Rox One-\n\nStep reaction buffer, 10 M forward and reverse primers, 10 units RiboSafe RNase inhibitor, and 5 L template RNA. Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification.", "Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification. Amount of the virus in the samples was quantified based on this standard curve. Analysis. Data statistical analysis was performed using SPSS 18.0. Data were represented as mean ± SE of three independent tests.", "Data were represented as mean ± SE of three independent tests. One-way ANOVA, Tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation.", "genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region (5 UTR), Open Reading Frames (ORFs) 1a and 1b, and 3 untranslated region (3 UTR) ( Table 1 ). The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex.", "The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. Both ends of the duplex TFOs were ligated with a linker sequence or clamps (C-C) to construct circular TFO RNA. Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO.", "Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE. Target Region.", "Target Region. The binding ability was determined using Electrophoretic Mobility Shift Assay (EMSA) [23] . The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region.", "The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region. The binding ability of different TFO RNAs (TFO1 to TFO5) against their target regions was determined by EMSA (Figure 2) . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band.", "TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band. This indicates the possession of triplex binding ability for all circular TFO RNAs, except TFO2. TFO RNA.", "TFO RNA. Study on the interaction and hybridization of TFO towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. As shown in supplementary Figure 1 (Table 3) . The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection.", "The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection. The results showed viral RNA genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with TFO1, TFO2, TFO3, TFO4, TFO5, and TFO7, respectively. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05).", "The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05). The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ( ≥ 0.05) ( Figure 3 ). The morphological changes of the cells were also captured 72 hours after transfection.", "The morphological changes of the cells were also captured 72 hours after transfection. The cells transfected with circular TFO1, TFO3, TFO4, and TFO5 appeared to be in good condition following virus inoculation, while the cells transfected with circular TFO2 and linear TFO3 and TFO4 showed visible cytopathic effect (CPE), the same as virus inoculated cells (supplementary Figure 2) . Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells.", "Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells. Hence, these results illustrated the capacity of circular TFO RNAs (except TFO2) to inhibit FIPV replication. Concentrations on FIPV Replication. Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs.", "Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs. The experimental conditions were identical to that of the previous experiment, except for TFO1 concentrations of 25 nM, 50 nM, 100 nM, and 500 nM. There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1.", "There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1. The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations ( Figure 4 ).", "However, no significant difference was detected in copy numbers from all of these concentrations ( Figure 4 ). The specificity of the TFO towards FIPV was tested, using TFO1 and TFO5, as the proper representatives of TFOs, on influenza A virus H1N1 New Jersey 8/76. The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary Figure 3 ).", "The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary Figure 3 ). Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift.", "EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. Binding of each circular TFO except circular TFO2 to its respective target forms a complex that migrates slower than unbound TFO. G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively.", "G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively. in the replication process [24] . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins [24] .", "Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins [24] . Hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit FIPV replication. Development of antiviral therapies against FIPV using siRNA [25] and viral protease inhibitors [26] Figure 4 : TFO1 dose-response study for inhibiting FIPV replication.", "Development of antiviral therapies against FIPV using siRNA [25] and viral protease inhibitors [26] Figure 4 : TFO1 dose-response study for inhibiting FIPV replication. The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction.", "50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction. Data are averages of 3 independent tests (mean ± SE). * Significantly different from FIPV-infected group. as potential new treatments against FIPV infection.", "* Significantly different from FIPV-infected group. as potential new treatments against FIPV infection. In this study, circular Triple Helix Forming Oligonucleotide (TFO) RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively.", "TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively. TFO3 to TFO5 targeted different regions of the ORF1a/1b on FIPV genome. Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis.", "Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis. All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO (Figure 1 ). The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate.", "The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate. EMSA was used to show the binding capability of each circular TFO towards the target region in the FIPV genome except for TFO2 which showed lack of formation of complex structure upon hybridization ( Figure 2) . The EMSA result also concurred with the antiviral study, where all circular TFOs (except TFO2) were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells (Figure 3 ).", "The EMSA result also concurred with the antiviral study, where all circular TFOs (except TFO2) were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells (Figure 3 ). However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions. Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems.", "Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems. Since, TFO1 shows the lowest value (Table 3) , the antiviral properties of this TFO were evaluated in doseresponse study. As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication.", "As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication. However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours [27, 28] .", "In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours [27, 28] . The above finding probably also explained the reason why no antiviral effect was detected when the transfection of the TFO was performed on virus-infected cells (data not shown). The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] .", "The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] . Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] .", "Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] . However, there are various factors that may limit the activity of TFOs in cells like intracellular degradation of the TFO and limited accessibility of the TFO to the target sites which can prevent triplex formation [31] . These findings may also explain the inability of the designed TFO1 to inhibit further virus replication in dose-response study (Figure 4) .", "These findings may also explain the inability of the designed TFO1 to inhibit further virus replication in dose-response study (Figure 4) . Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] .", "However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] . Recently, McDonagh et al. [25] developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells.", "[25] developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells. The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus [13] and TFO to inhibit the transcription of 1(I) collagen in rat fibroblasts [14] , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] .", "In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] . These studies indicated the flexibility of using TFO-based oligonucleotides as a potential molecular-based therapy. In this study, we demonstrated short circular TFO RNAs between 28 and 34 mers (Table 1) , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome.", "In this study, we demonstrated short circular TFO RNAs between 28 and 34 mers (Table 1) , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome. All designed circular TFOs (except TFO2) showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication.", "The TFOs that formed triplex structures showed antiviral effects towards FIPV replication. The reason why TFO2 failed to show any interaction with the target region or antiviral activity is probably due to the length of TFO2 (i.e., 24 mers), which might be insufficient to a triplex formation upon hybridization (Figure 2 ), be effective enough to suppress viral RNA transcription, and eventually inhibit virus replication. Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out.", "Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. In vitro antiviral study which showed no antiviral property for unrelated TFO (TFO7) and also inability of circular TFO1 and TFO5 to inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity. In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats.", "In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats. However, further studies on TFO specificity, actual mechanism of circular TFO RNA in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future." ]

[ "Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective.", "Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells.", "RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay.", "The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Text: Feline Infectious Peritonitis Virus (FIPV) is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome.", "Text: Feline Infectious Peritonitis Virus (FIPV) is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome. FIPV is grouped as feline coronavirus (FCoV), under the family Coronaviridae. FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus (FECV), a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV [1] .", "FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus (FECV), a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV [1] . The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory.", "Two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory. Internal mutation theory stated that the development of FIP is due to the exposure of cat to variants of FCoV which have been mutated by gaining the ability to replicate within the macrophages [2] , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population [3] . Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces [4] .", "Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces [4] . About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease [4] . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV.", "The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV. Vaccination against FIPV with an attenuated, temperature-sensitive strain of type II FIPV induces low antibody titre in kittens that have not been exposed to FCoV. However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] .", "However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] . In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement [1] . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] .", "Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] . However, those treatments were ineffective. Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection.", "Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection. Triple Helix Forming Oligonucleotide (TFO) is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA [9] . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] .", "Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] . Hence, TFO has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes [11] [12] [13] [14] [15] [16] . The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication.", "The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication. serotype II strain WSU 79-1146 (ATCC no. VR-1777) was grown in CRFK cells. A serial 10-fold dilution of FIPV was prepared from the working stock.", "A serial 10-fold dilution of FIPV was prepared from the working stock. Confluent 96-well plate was inoculated with 100 L of each virus dilution/well. The plate was incubated in a humidified incubator at 37 ∘ C, 5% CO 2 . Cytopathic effects (CPE) development was observed.", "Cytopathic effects (CPE) development was observed. The results were recorded after 72 hours and the virus tissue culture infective dose 50 (TCID 50 ) was calculated using Reed and Muench's method [17] . Oligonucleotide RNA.", "Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides (TFOs) were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 (Accession no: AY994055) [18] . TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed ( Table 1 ).", "TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed ( Table 1 ). The specificity of the TFOs was identified using BLAST search in the NCBI database. The designed linear TFOs were synthesized by Dharmacon Research (USA), whereby the 5 and 3 ends of the linear TFOs were modified with phosphate (PO 4 ) group and hydroxide (OH) group, respectively.", "The designed linear TFOs were synthesized by Dharmacon Research (USA), whereby the 5 and 3 ends of the linear TFOs were modified with phosphate (PO 4 ) group and hydroxide (OH) group, respectively. These modifications were necessary for the circularization of linear TFO. The process of circularization, using the T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs Inc., England), was carried out according to the manufacturer's protocol.", "The process of circularization, using the T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs Inc., England), was carried out according to the manufacturer's protocol. After ligation, the circular TFO RNAs were recovered by ethanol precipitation and the purity of the circular TFO RNAs was measured using spectrophotometer. Denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification.", "Denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. Then, the gel was prerun at 20 to 40 V for 45 minutes.", "Then, the gel was prerun at 20 to 40 V for 45 minutes. Five L of TFO RNA mixed with 5 L of urea loading buffer was heated at 92 ∘ C for 2 minutes and immediately chilled on ice. It was run on the gel at 200 V for 45 minutes.", "It was run on the gel at 200 V for 45 minutes. Finally, the gel was stained with ethidium bromide (Sigma, USA) and viewed with a Bio-Rad Gel Doc XR system (CA, USA). (EMSA) .", "(EMSA) . The target regions of the FIPV genome were synthesized by Dharmacon Research (USA) ( Table 1) . Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours.", "Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours. The sample was run on 15% native polyacrylamide gel at 80 V, in cool condition. The stained gel was viewed by a Bio-Rad Gel Doc XR system.", "The stained gel was viewed by a Bio-Rad Gel Doc XR system. Regions. The binding strength was measured using a nano Isothermal Titration Calorimeter (ITC) (TA instruments, Newcastle, UK).", "The binding strength was measured using a nano Isothermal Titration Calorimeter (ITC) (TA instruments, Newcastle, UK). The RNA sample mixtures, consisting of circular TFOs (0.0002 mM), were incubated with their respective synthetic target regions (0.015 mM) using 1X binding buffer as the diluent. The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections.", "The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer. This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection.", "This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. One hundred nM of TFO RNAs was separately transfected into the CRFK cells using a HiPerFect Transfection Reagent (Qiagen, Germany), as per the manufacturer's protocol. The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours.", "The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C (100 L/well). Finally, the viral inoculum was replaced by fresh maintenance media (MEM containing 1% FBS and 1% pen/strep).", "Finally, the viral inoculum was replaced by fresh maintenance media (MEM containing 1% FBS and 1% pen/strep). Virus-infected and uninfected cells were maintained as positive and negative controls, respectively. The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction.", "The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction. Inhibition. Different concentrations of circular TFO1 RNA (25 nM, 50 nM, 100 nM, and 500 nM) were transfected into CRFK cells.", "Different concentrations of circular TFO1 RNA (25 nM, 50 nM, 100 nM, and 500 nM) were transfected into CRFK cells. The plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ C with 5% CO2. The cells were processed as described above.", "The cells were processed as described above. Madin-Darby Canine Kidney (MDCK) cell (ATCC no. CCL-34), at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. Transfection was performed the same as before.", "Transfection was performed the same as before. One hundred nM of circular TFO RNA was transfected into MDCK cells. Following 6 hours \n\nORF1a/1b and 530-541\n\nORF1a/1b and 7399-7411\n\nORF1a/1b and 14048-14061\n\n- * Highlighted in bold indicated the binding region. * * Unrelated circular TFO. [20, 21] , respectively.", "* * Unrelated circular TFO. [20, 21] , respectively. The reverse transcriptase quantitative real-time PCR (RT-qPCR) was performed using a Bio-Rad CFX96 real-time system (BioRad, USA).", "The reverse transcriptase quantitative real-time PCR (RT-qPCR) was performed using a Bio-Rad CFX96 real-time system (BioRad, USA). The reaction was amplified in a final volume of 25 L using a SensiMix SYBR No-ROX One-Step Kit (Bioline, UK), which consisted of 12.5 L 2X SensiMix SYBR No-Rox One-\n\nStep reaction buffer, 10 M forward and reverse primers, 10 units RiboSafe RNase inhibitor, and 5 L template RNA. Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification.", "Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification. Amount of the virus in the samples was quantified based on this standard curve. Analysis. Data statistical analysis was performed using SPSS 18.0. Data were represented as mean ± SE of three independent tests.", "Data were represented as mean ± SE of three independent tests. One-way ANOVA, Tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation.", "genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region (5 UTR), Open Reading Frames (ORFs) 1a and 1b, and 3 untranslated region (3 UTR) ( Table 1 ). The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex.", "The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. Both ends of the duplex TFOs were ligated with a linker sequence or clamps (C-C) to construct circular TFO RNA. Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO.", "Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE. Target Region.", "Target Region. The binding ability was determined using Electrophoretic Mobility Shift Assay (EMSA) [23] . The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region.", "The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region. The binding ability of different TFO RNAs (TFO1 to TFO5) against their target regions was determined by EMSA (Figure 2) . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band.", "TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band. This indicates the possession of triplex binding ability for all circular TFO RNAs, except TFO2. TFO RNA.", "TFO RNA. Study on the interaction and hybridization of TFO towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. As shown in supplementary Figure 1 (Table 3) . The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection.", "The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection. The results showed viral RNA genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with TFO1, TFO2, TFO3, TFO4, TFO5, and TFO7, respectively. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05).", "The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05). The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ( ≥ 0.05) ( Figure 3 ). The morphological changes of the cells were also captured 72 hours after transfection.", "The morphological changes of the cells were also captured 72 hours after transfection. The cells transfected with circular TFO1, TFO3, TFO4, and TFO5 appeared to be in good condition following virus inoculation, while the cells transfected with circular TFO2 and linear TFO3 and TFO4 showed visible cytopathic effect (CPE), the same as virus inoculated cells (supplementary Figure 2) . Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells.", "Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells. Hence, these results illustrated the capacity of circular TFO RNAs (except TFO2) to inhibit FIPV replication. Concentrations on FIPV Replication. Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs.", "Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs. The experimental conditions were identical to that of the previous experiment, except for TFO1 concentrations of 25 nM, 50 nM, 100 nM, and 500 nM. There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1.", "There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1. The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations ( Figure 4 ).", "However, no significant difference was detected in copy numbers from all of these concentrations ( Figure 4 ). The specificity of the TFO towards FIPV was tested, using TFO1 and TFO5, as the proper representatives of TFOs, on influenza A virus H1N1 New Jersey 8/76. The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary Figure 3 ).", "The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary Figure 3 ). Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift.", "EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. Binding of each circular TFO except circular TFO2 to its respective target forms a complex that migrates slower than unbound TFO. G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively.", "G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively. in the replication process [24] . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins [24] .", "Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins [24] . Hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit FIPV replication. Development of antiviral therapies against FIPV using siRNA [25] and viral protease inhibitors [26] Figure 4 : TFO1 dose-response study for inhibiting FIPV replication.", "Development of antiviral therapies against FIPV using siRNA [25] and viral protease inhibitors [26] Figure 4 : TFO1 dose-response study for inhibiting FIPV replication. The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction.", "50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction. Data are averages of 3 independent tests (mean ± SE). * Significantly different from FIPV-infected group. as potential new treatments against FIPV infection.", "* Significantly different from FIPV-infected group. as potential new treatments against FIPV infection. In this study, circular Triple Helix Forming Oligonucleotide (TFO) RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively.", "TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively. TFO3 to TFO5 targeted different regions of the ORF1a/1b on FIPV genome. Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis.", "Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis. All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO (Figure 1 ). The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate.", "The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate. EMSA was used to show the binding capability of each circular TFO towards the target region in the FIPV genome except for TFO2 which showed lack of formation of complex structure upon hybridization ( Figure 2) . The EMSA result also concurred with the antiviral study, where all circular TFOs (except TFO2) were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells (Figure 3 ).", "The EMSA result also concurred with the antiviral study, where all circular TFOs (except TFO2) were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells (Figure 3 ). However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions. Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems.", "Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems. Since, TFO1 shows the lowest value (Table 3) , the antiviral properties of this TFO were evaluated in doseresponse study. As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication.", "As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication. However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours [27, 28] .", "In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours [27, 28] . The above finding probably also explained the reason why no antiviral effect was detected when the transfection of the TFO was performed on virus-infected cells (data not shown). The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] .", "The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] . Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] .", "Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] . However, there are various factors that may limit the activity of TFOs in cells like intracellular degradation of the TFO and limited accessibility of the TFO to the target sites which can prevent triplex formation [31] . These findings may also explain the inability of the designed TFO1 to inhibit further virus replication in dose-response study (Figure 4) .", "These findings may also explain the inability of the designed TFO1 to inhibit further virus replication in dose-response study (Figure 4) . Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] .", "However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] . Recently, McDonagh et al. [25] developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells.", "[25] developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells. The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus [13] and TFO to inhibit the transcription of 1(I) collagen in rat fibroblasts [14] , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] .", "In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] . These studies indicated the flexibility of using TFO-based oligonucleotides as a potential molecular-based therapy. In this study, we demonstrated short circular TFO RNAs between 28 and 34 mers (Table 1) , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome.", "In this study, we demonstrated short circular TFO RNAs between 28 and 34 mers (Table 1) , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome. All designed circular TFOs (except TFO2) showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication.", "The TFOs that formed triplex structures showed antiviral effects towards FIPV replication. The reason why TFO2 failed to show any interaction with the target region or antiviral activity is probably due to the length of TFO2 (i.e., 24 mers), which might be insufficient to a triplex formation upon hybridization (Figure 2 ), be effective enough to suppress viral RNA transcription, and eventually inhibit virus replication. Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out.", "Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. In vitro antiviral study which showed no antiviral property for unrelated TFO (TFO7) and also inability of circular TFO1 and TFO5 to inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity. In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats.", "In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats. However, further studies on TFO specificity, actual mechanism of circular TFO RNA in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future." ]

[ "Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective.", "Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells.", "RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay.", "The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Text: Feline Infectious Peritonitis Virus (FIPV) is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome.", "Text: Feline Infectious Peritonitis Virus (FIPV) is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome. FIPV is grouped as feline coronavirus (FCoV), under the family Coronaviridae. FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus (FECV), a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV [1] .", "FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus (FECV), a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV [1] . The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory.", "Two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory. Internal mutation theory stated that the development of FIP is due to the exposure of cat to variants of FCoV which have been mutated by gaining the ability to replicate within the macrophages [2] , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population [3] . Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces [4] .", "Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces [4] . About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease [4] . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV.", "The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV. Vaccination against FIPV with an attenuated, temperature-sensitive strain of type II FIPV induces low antibody titre in kittens that have not been exposed to FCoV. However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] .", "However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] . In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement [1] . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] .", "Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] . However, those treatments were ineffective. Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection.", "Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection. Triple Helix Forming Oligonucleotide (TFO) is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA [9] . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] .", "Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] . Hence, TFO has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes [11] [12] [13] [14] [15] [16] . The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication.", "The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication. serotype II strain WSU 79-1146 (ATCC no. VR-1777) was grown in CRFK cells. A serial 10-fold dilution of FIPV was prepared from the working stock.", "A serial 10-fold dilution of FIPV was prepared from the working stock. Confluent 96-well plate was inoculated with 100 L of each virus dilution/well. The plate was incubated in a humidified incubator at 37 ∘ C, 5% CO 2 . Cytopathic effects (CPE) development was observed.", "Cytopathic effects (CPE) development was observed. The results were recorded after 72 hours and the virus tissue culture infective dose 50 (TCID 50 ) was calculated using Reed and Muench's method [17] . Oligonucleotide RNA.", "Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides (TFOs) were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 (Accession no: AY994055) [18] . TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed ( Table 1 ).", "TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed ( Table 1 ). The specificity of the TFOs was identified using BLAST search in the NCBI database. The designed linear TFOs were synthesized by Dharmacon Research (USA), whereby the 5 and 3 ends of the linear TFOs were modified with phosphate (PO 4 ) group and hydroxide (OH) group, respectively.", "The designed linear TFOs were synthesized by Dharmacon Research (USA), whereby the 5 and 3 ends of the linear TFOs were modified with phosphate (PO 4 ) group and hydroxide (OH) group, respectively. These modifications were necessary for the circularization of linear TFO. The process of circularization, using the T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs Inc., England), was carried out according to the manufacturer's protocol.", "The process of circularization, using the T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs Inc., England), was carried out according to the manufacturer's protocol. After ligation, the circular TFO RNAs were recovered by ethanol precipitation and the purity of the circular TFO RNAs was measured using spectrophotometer. Denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification.", "Denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. Then, the gel was prerun at 20 to 40 V for 45 minutes.", "Then, the gel was prerun at 20 to 40 V for 45 minutes. Five L of TFO RNA mixed with 5 L of urea loading buffer was heated at 92 ∘ C for 2 minutes and immediately chilled on ice. It was run on the gel at 200 V for 45 minutes.", "It was run on the gel at 200 V for 45 minutes. Finally, the gel was stained with ethidium bromide (Sigma, USA) and viewed with a Bio-Rad Gel Doc XR system (CA, USA). (EMSA) .", "(EMSA) . The target regions of the FIPV genome were synthesized by Dharmacon Research (USA) ( Table 1) . Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours.", "Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours. The sample was run on 15% native polyacrylamide gel at 80 V, in cool condition. The stained gel was viewed by a Bio-Rad Gel Doc XR system.", "The stained gel was viewed by a Bio-Rad Gel Doc XR system. Regions. The binding strength was measured using a nano Isothermal Titration Calorimeter (ITC) (TA instruments, Newcastle, UK).", "The binding strength was measured using a nano Isothermal Titration Calorimeter (ITC) (TA instruments, Newcastle, UK). The RNA sample mixtures, consisting of circular TFOs (0.0002 mM), were incubated with their respective synthetic target regions (0.015 mM) using 1X binding buffer as the diluent. The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections.", "The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer. This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection.", "This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. One hundred nM of TFO RNAs was separately transfected into the CRFK cells using a HiPerFect Transfection Reagent (Qiagen, Germany), as per the manufacturer's protocol. The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours.", "The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C (100 L/well). Finally, the viral inoculum was replaced by fresh maintenance media (MEM containing 1% FBS and 1% pen/strep).", "Finally, the viral inoculum was replaced by fresh maintenance media (MEM containing 1% FBS and 1% pen/strep). Virus-infected and uninfected cells were maintained as positive and negative controls, respectively. The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction.", "The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction. Inhibition. Different concentrations of circular TFO1 RNA (25 nM, 50 nM, 100 nM, and 500 nM) were transfected into CRFK cells.", "Different concentrations of circular TFO1 RNA (25 nM, 50 nM, 100 nM, and 500 nM) were transfected into CRFK cells. The plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ C with 5% CO2. The cells were processed as described above.", "The cells were processed as described above. Madin-Darby Canine Kidney (MDCK) cell (ATCC no. CCL-34), at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. Transfection was performed the same as before.", "Transfection was performed the same as before. One hundred nM of circular TFO RNA was transfected into MDCK cells. Following 6 hours \n\nORF1a/1b and 530-541\n\nORF1a/1b and 7399-7411\n\nORF1a/1b and 14048-14061\n\n- * Highlighted in bold indicated the binding region. * * Unrelated circular TFO. [20, 21] , respectively.", "* * Unrelated circular TFO. [20, 21] , respectively. The reverse transcriptase quantitative real-time PCR (RT-qPCR) was performed using a Bio-Rad CFX96 real-time system (BioRad, USA).", "The reverse transcriptase quantitative real-time PCR (RT-qPCR) was performed using a Bio-Rad CFX96 real-time system (BioRad, USA). The reaction was amplified in a final volume of 25 L using a SensiMix SYBR No-ROX One-Step Kit (Bioline, UK), which consisted of 12.5 L 2X SensiMix SYBR No-Rox One-\n\nStep reaction buffer, 10 M forward and reverse primers, 10 units RiboSafe RNase inhibitor, and 5 L template RNA. Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification.", "Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification. Amount of the virus in the samples was quantified based on this standard curve. Analysis. Data statistical analysis was performed using SPSS 18.0. Data were represented as mean ± SE of three independent tests.", "Data were represented as mean ± SE of three independent tests. One-way ANOVA, Tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation.", "genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region (5 UTR), Open Reading Frames (ORFs) 1a and 1b, and 3 untranslated region (3 UTR) ( Table 1 ). The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex.", "The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. Both ends of the duplex TFOs were ligated with a linker sequence or clamps (C-C) to construct circular TFO RNA. Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO.", "Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE. Target Region.", "Target Region. The binding ability was determined using Electrophoretic Mobility Shift Assay (EMSA) [23] . The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region.", "The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region. The binding ability of different TFO RNAs (TFO1 to TFO5) against their target regions was determined by EMSA (Figure 2) . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band.", "TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band. This indicates the possession of triplex binding ability for all circular TFO RNAs, except TFO2. TFO RNA.", "TFO RNA. Study on the interaction and hybridization of TFO towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. As shown in supplementary Figure 1 (Table 3) . The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection.", "The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection. The results showed viral RNA genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with TFO1, TFO2, TFO3, TFO4, TFO5, and TFO7, respectively. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05).", "The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05). The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ( ≥ 0.05) ( Figure 3 ). The morphological changes of the cells were also captured 72 hours after transfection.", "The morphological changes of the cells were also captured 72 hours after transfection. The cells transfected with circular TFO1, TFO3, TFO4, and TFO5 appeared to be in good condition following virus inoculation, while the cells transfected with circular TFO2 and linear TFO3 and TFO4 showed visible cytopathic effect (CPE), the same as virus inoculated cells (supplementary Figure 2) . Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells.", "Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells. Hence, these results illustrated the capacity of circular TFO RNAs (except TFO2) to inhibit FIPV replication. Concentrations on FIPV Replication. Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs.", "Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs. The experimental conditions were identical to that of the previous experiment, except for TFO1 concentrations of 25 nM, 50 nM, 100 nM, and 500 nM. There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1.", "There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1. The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations ( Figure 4 ).", "However, no significant difference was detected in copy numbers from all of these concentrations ( Figure 4 ). The specificity of the TFO towards FIPV was tested, using TFO1 and TFO5, as the proper representatives of TFOs, on influenza A virus H1N1 New Jersey 8/76. The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary Figure 3 ).", "The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary Figure 3 ). Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift.", "EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. Binding of each circular TFO except circular TFO2 to its respective target forms a complex that migrates slower than unbound TFO. G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively.", "G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively. in the replication process [24] . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins [24] .", "Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins [24] . Hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit FIPV replication. Development of antiviral therapies against FIPV using siRNA [25] and viral protease inhibitors [26] Figure 4 : TFO1 dose-response study for inhibiting FIPV replication.", "Development of antiviral therapies against FIPV using siRNA [25] and viral protease inhibitors [26] Figure 4 : TFO1 dose-response study for inhibiting FIPV replication. The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction.", "50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction. Data are averages of 3 independent tests (mean ± SE). * Significantly different from FIPV-infected group. as potential new treatments against FIPV infection.", "* Significantly different from FIPV-infected group. as potential new treatments against FIPV infection. In this study, circular Triple Helix Forming Oligonucleotide (TFO) RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively.", "TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively. TFO3 to TFO5 targeted different regions of the ORF1a/1b on FIPV genome. Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis.", "Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis. All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO (Figure 1 ). The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate.", "The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate. EMSA was used to show the binding capability of each circular TFO towards the target region in the FIPV genome except for TFO2 which showed lack of formation of complex structure upon hybridization ( Figure 2) . The EMSA result also concurred with the antiviral study, where all circular TFOs (except TFO2) were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells (Figure 3 ).", "The EMSA result also concurred with the antiviral study, where all circular TFOs (except TFO2) were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells (Figure 3 ). However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions. Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems.", "Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems. Since, TFO1 shows the lowest value (Table 3) , the antiviral properties of this TFO were evaluated in doseresponse study. As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication.", "As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication. However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours [27, 28] .", "In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours [27, 28] . The above finding probably also explained the reason why no antiviral effect was detected when the transfection of the TFO was performed on virus-infected cells (data not shown). The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] .", "The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] . Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] .", "Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] . However, there are various factors that may limit the activity of TFOs in cells like intracellular degradation of the TFO and limited accessibility of the TFO to the target sites which can prevent triplex formation [31] . These findings may also explain the inability of the designed TFO1 to inhibit further virus replication in dose-response study (Figure 4) .", "These findings may also explain the inability of the designed TFO1 to inhibit further virus replication in dose-response study (Figure 4) . Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] .", "However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] . Recently, McDonagh et al. [25] developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells.", "[25] developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells. The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus [13] and TFO to inhibit the transcription of 1(I) collagen in rat fibroblasts [14] , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] .", "In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] . These studies indicated the flexibility of using TFO-based oligonucleotides as a potential molecular-based therapy. In this study, we demonstrated short circular TFO RNAs between 28 and 34 mers (Table 1) , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome.", "In this study, we demonstrated short circular TFO RNAs between 28 and 34 mers (Table 1) , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome. All designed circular TFOs (except TFO2) showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication.", "The TFOs that formed triplex structures showed antiviral effects towards FIPV replication. The reason why TFO2 failed to show any interaction with the target region or antiviral activity is probably due to the length of TFO2 (i.e., 24 mers), which might be insufficient to a triplex formation upon hybridization (Figure 2 ), be effective enough to suppress viral RNA transcription, and eventually inhibit virus replication. Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out.", "Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. In vitro antiviral study which showed no antiviral property for unrelated TFO (TFO7) and also inability of circular TFO1 and TFO5 to inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity. In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats.", "In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats. However, further studies on TFO specificity, actual mechanism of circular TFO RNA in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future." ]

[ "Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective.", "Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells.", "RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay.", "The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Text: Feline Infectious Peritonitis Virus (FIPV) is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome.", "Text: Feline Infectious Peritonitis Virus (FIPV) is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome. FIPV is grouped as feline coronavirus (FCoV), under the family Coronaviridae. FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus (FECV), a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV [1] .", "FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus (FECV), a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV [1] . The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory.", "Two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory. Internal mutation theory stated that the development of FIP is due to the exposure of cat to variants of FCoV which have been mutated by gaining the ability to replicate within the macrophages [2] , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population [3] . Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces [4] .", "Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces [4] . About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease [4] . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV.", "The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV. Vaccination against FIPV with an attenuated, temperature-sensitive strain of type II FIPV induces low antibody titre in kittens that have not been exposed to FCoV. However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] .", "However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] . In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement [1] . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] .", "Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] . However, those treatments were ineffective. Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection.", "Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection. Triple Helix Forming Oligonucleotide (TFO) is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA [9] . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] .", "Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] . Hence, TFO has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes [11] [12] [13] [14] [15] [16] . The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication.", "The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication. serotype II strain WSU 79-1146 (ATCC no. VR-1777) was grown in CRFK cells. A serial 10-fold dilution of FIPV was prepared from the working stock.", "A serial 10-fold dilution of FIPV was prepared from the working stock. Confluent 96-well plate was inoculated with 100 L of each virus dilution/well. The plate was incubated in a humidified incubator at 37 ∘ C, 5% CO 2 . Cytopathic effects (CPE) development was observed.", "Cytopathic effects (CPE) development was observed. The results were recorded after 72 hours and the virus tissue culture infective dose 50 (TCID 50 ) was calculated using Reed and Muench's method [17] . Oligonucleotide RNA.", "Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides (TFOs) were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 (Accession no: AY994055) [18] . TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed ( Table 1 ).", "TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed ( Table 1 ). The specificity of the TFOs was identified using BLAST search in the NCBI database. The designed linear TFOs were synthesized by Dharmacon Research (USA), whereby the 5 and 3 ends of the linear TFOs were modified with phosphate (PO 4 ) group and hydroxide (OH) group, respectively.", "The designed linear TFOs were synthesized by Dharmacon Research (USA), whereby the 5 and 3 ends of the linear TFOs were modified with phosphate (PO 4 ) group and hydroxide (OH) group, respectively. These modifications were necessary for the circularization of linear TFO. The process of circularization, using the T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs Inc., England), was carried out according to the manufacturer's protocol.", "The process of circularization, using the T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs Inc., England), was carried out according to the manufacturer's protocol. After ligation, the circular TFO RNAs were recovered by ethanol precipitation and the purity of the circular TFO RNAs was measured using spectrophotometer. Denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification.", "Denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. Then, the gel was prerun at 20 to 40 V for 45 minutes.", "Then, the gel was prerun at 20 to 40 V for 45 minutes. Five L of TFO RNA mixed with 5 L of urea loading buffer was heated at 92 ∘ C for 2 minutes and immediately chilled on ice. It was run on the gel at 200 V for 45 minutes.", "It was run on the gel at 200 V for 45 minutes. Finally, the gel was stained with ethidium bromide (Sigma, USA) and viewed with a Bio-Rad Gel Doc XR system (CA, USA). (EMSA) .", "(EMSA) . The target regions of the FIPV genome were synthesized by Dharmacon Research (USA) ( Table 1) . Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours.", "Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours. The sample was run on 15% native polyacrylamide gel at 80 V, in cool condition. The stained gel was viewed by a Bio-Rad Gel Doc XR system.", "The stained gel was viewed by a Bio-Rad Gel Doc XR system. Regions. The binding strength was measured using a nano Isothermal Titration Calorimeter (ITC) (TA instruments, Newcastle, UK).", "The binding strength was measured using a nano Isothermal Titration Calorimeter (ITC) (TA instruments, Newcastle, UK). The RNA sample mixtures, consisting of circular TFOs (0.0002 mM), were incubated with their respective synthetic target regions (0.015 mM) using 1X binding buffer as the diluent. The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections.", "The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer. This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection.", "This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. One hundred nM of TFO RNAs was separately transfected into the CRFK cells using a HiPerFect Transfection Reagent (Qiagen, Germany), as per the manufacturer's protocol. The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours.", "The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C (100 L/well). Finally, the viral inoculum was replaced by fresh maintenance media (MEM containing 1% FBS and 1% pen/strep).", "Finally, the viral inoculum was replaced by fresh maintenance media (MEM containing 1% FBS and 1% pen/strep). Virus-infected and uninfected cells were maintained as positive and negative controls, respectively. The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction.", "The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction. Inhibition. Different concentrations of circular TFO1 RNA (25 nM, 50 nM, 100 nM, and 500 nM) were transfected into CRFK cells.", "Different concentrations of circular TFO1 RNA (25 nM, 50 nM, 100 nM, and 500 nM) were transfected into CRFK cells. The plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ C with 5% CO2. The cells were processed as described above.", "The cells were processed as described above. Madin-Darby Canine Kidney (MDCK) cell (ATCC no. CCL-34), at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. Transfection was performed the same as before.", "Transfection was performed the same as before. One hundred nM of circular TFO RNA was transfected into MDCK cells. Following 6 hours \n\nORF1a/1b and 530-541\n\nORF1a/1b and 7399-7411\n\nORF1a/1b and 14048-14061\n\n- * Highlighted in bold indicated the binding region. * * Unrelated circular TFO. [20, 21] , respectively.", "* * Unrelated circular TFO. [20, 21] , respectively. The reverse transcriptase quantitative real-time PCR (RT-qPCR) was performed using a Bio-Rad CFX96 real-time system (BioRad, USA).", "The reverse transcriptase quantitative real-time PCR (RT-qPCR) was performed using a Bio-Rad CFX96 real-time system (BioRad, USA). The reaction was amplified in a final volume of 25 L using a SensiMix SYBR No-ROX One-Step Kit (Bioline, UK), which consisted of 12.5 L 2X SensiMix SYBR No-Rox One-\n\nStep reaction buffer, 10 M forward and reverse primers, 10 units RiboSafe RNase inhibitor, and 5 L template RNA. Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification.", "Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification. Amount of the virus in the samples was quantified based on this standard curve. Analysis. Data statistical analysis was performed using SPSS 18.0. Data were represented as mean ± SE of three independent tests.", "Data were represented as mean ± SE of three independent tests. One-way ANOVA, Tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation.", "genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region (5 UTR), Open Reading Frames (ORFs) 1a and 1b, and 3 untranslated region (3 UTR) ( Table 1 ). The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex.", "The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. Both ends of the duplex TFOs were ligated with a linker sequence or clamps (C-C) to construct circular TFO RNA. Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO.", "Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE. Target Region.", "Target Region. The binding ability was determined using Electrophoretic Mobility Shift Assay (EMSA) [23] . The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region.", "The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region. The binding ability of different TFO RNAs (TFO1 to TFO5) against their target regions was determined by EMSA (Figure 2) . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band.", "TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band. This indicates the possession of triplex binding ability for all circular TFO RNAs, except TFO2. TFO RNA.", "TFO RNA. Study on the interaction and hybridization of TFO towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. As shown in supplementary Figure 1 (Table 3) . The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection.", "The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection. The results showed viral RNA genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with TFO1, TFO2, TFO3, TFO4, TFO5, and TFO7, respectively. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05).", "The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05). The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ( ≥ 0.05) ( Figure 3 ). The morphological changes of the cells were also captured 72 hours after transfection.", "The morphological changes of the cells were also captured 72 hours after transfection. The cells transfected with circular TFO1, TFO3, TFO4, and TFO5 appeared to be in good condition following virus inoculation, while the cells transfected with circular TFO2 and linear TFO3 and TFO4 showed visible cytopathic effect (CPE), the same as virus inoculated cells (supplementary Figure 2) . Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells.", "Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells. Hence, these results illustrated the capacity of circular TFO RNAs (except TFO2) to inhibit FIPV replication. Concentrations on FIPV Replication. Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs.", "Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs. The experimental conditions were identical to that of the previous experiment, except for TFO1 concentrations of 25 nM, 50 nM, 100 nM, and 500 nM. There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1.", "There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1. The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations ( Figure 4 ).", "However, no significant difference was detected in copy numbers from all of these concentrations ( Figure 4 ). The specificity of the TFO towards FIPV was tested, using TFO1 and TFO5, as the proper representatives of TFOs, on influenza A virus H1N1 New Jersey 8/76. The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary Figure 3 ).", "The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary Figure 3 ). Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift.", "EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. Binding of each circular TFO except circular TFO2 to its respective target forms a complex that migrates slower than unbound TFO. G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively.", "G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively. in the replication process [24] . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins [24] .", "Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins [24] . Hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit FIPV replication. Development of antiviral therapies against FIPV using siRNA [25] and viral protease inhibitors [26] Figure 4 : TFO1 dose-response study for inhibiting FIPV replication.", "Development of antiviral therapies against FIPV using siRNA [25] and viral protease inhibitors [26] Figure 4 : TFO1 dose-response study for inhibiting FIPV replication. The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction.", "50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction. Data are averages of 3 independent tests (mean ± SE). * Significantly different from FIPV-infected group. as potential new treatments against FIPV infection.", "* Significantly different from FIPV-infected group. as potential new treatments against FIPV infection. In this study, circular Triple Helix Forming Oligonucleotide (TFO) RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively.", "TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively. TFO3 to TFO5 targeted different regions of the ORF1a/1b on FIPV genome. Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis.", "Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis. All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO (Figure 1 ). The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate.", "The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate. EMSA was used to show the binding capability of each circular TFO towards the target region in the FIPV genome except for TFO2 which showed lack of formation of complex structure upon hybridization ( Figure 2) . The EMSA result also concurred with the antiviral study, where all circular TFOs (except TFO2) were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells (Figure 3 ).", "The EMSA result also concurred with the antiviral study, where all circular TFOs (except TFO2) were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells (Figure 3 ). However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions. Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems.", "Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems. Since, TFO1 shows the lowest value (Table 3) , the antiviral properties of this TFO were evaluated in doseresponse study. As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication.", "As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication. However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours [27, 28] .", "In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours [27, 28] . The above finding probably also explained the reason why no antiviral effect was detected when the transfection of the TFO was performed on virus-infected cells (data not shown). The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] .", "The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] . Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] .", "Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] . However, there are various factors that may limit the activity of TFOs in cells like intracellular degradation of the TFO and limited accessibility of the TFO to the target sites which can prevent triplex formation [31] . These findings may also explain the inability of the designed TFO1 to inhibit further virus replication in dose-response study (Figure 4) .", "These findings may also explain the inability of the designed TFO1 to inhibit further virus replication in dose-response study (Figure 4) . Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] .", "However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] . Recently, McDonagh et al. [25] developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells.", "[25] developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells. The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus [13] and TFO to inhibit the transcription of 1(I) collagen in rat fibroblasts [14] , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] .", "In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] . These studies indicated the flexibility of using TFO-based oligonucleotides as a potential molecular-based therapy. In this study, we demonstrated short circular TFO RNAs between 28 and 34 mers (Table 1) , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome.", "In this study, we demonstrated short circular TFO RNAs between 28 and 34 mers (Table 1) , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome. All designed circular TFOs (except TFO2) showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication.", "The TFOs that formed triplex structures showed antiviral effects towards FIPV replication. The reason why TFO2 failed to show any interaction with the target region or antiviral activity is probably due to the length of TFO2 (i.e., 24 mers), which might be insufficient to a triplex formation upon hybridization (Figure 2 ), be effective enough to suppress viral RNA transcription, and eventually inhibit virus replication. Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out.", "Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. In vitro antiviral study which showed no antiviral property for unrelated TFO (TFO7) and also inability of circular TFO1 and TFO5 to inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity. In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats.", "In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats. However, further studies on TFO specificity, actual mechanism of circular TFO RNA in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future." ]

[ "Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective.", "Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells.", "RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay.", "The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Text: Feline Infectious Peritonitis Virus (FIPV) is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome.", "Text: Feline Infectious Peritonitis Virus (FIPV) is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome. FIPV is grouped as feline coronavirus (FCoV), under the family Coronaviridae. FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus (FECV), a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV [1] .", "FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus (FECV), a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV [1] . The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory.", "Two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory. Internal mutation theory stated that the development of FIP is due to the exposure of cat to variants of FCoV which have been mutated by gaining the ability to replicate within the macrophages [2] , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population [3] . Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces [4] .", "Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces [4] . About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease [4] . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV.", "The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV. Vaccination against FIPV with an attenuated, temperature-sensitive strain of type II FIPV induces low antibody titre in kittens that have not been exposed to FCoV. However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] .", "However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] . In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement [1] . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] .", "Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] . However, those treatments were ineffective. Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection.", "Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection. Triple Helix Forming Oligonucleotide (TFO) is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA [9] . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] .", "Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] . Hence, TFO has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes [11] [12] [13] [14] [15] [16] . The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication.", "The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication. serotype II strain WSU 79-1146 (ATCC no. VR-1777) was grown in CRFK cells. A serial 10-fold dilution of FIPV was prepared from the working stock.", "A serial 10-fold dilution of FIPV was prepared from the working stock. Confluent 96-well plate was inoculated with 100 L of each virus dilution/well. The plate was incubated in a humidified incubator at 37 ∘ C, 5% CO 2 . Cytopathic effects (CPE) development was observed.", "Cytopathic effects (CPE) development was observed. The results were recorded after 72 hours and the virus tissue culture infective dose 50 (TCID 50 ) was calculated using Reed and Muench's method [17] . Oligonucleotide RNA.", "Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides (TFOs) were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 (Accession no: AY994055) [18] . TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed ( Table 1 ).", "TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed ( Table 1 ). The specificity of the TFOs was identified using BLAST search in the NCBI database. The designed linear TFOs were synthesized by Dharmacon Research (USA), whereby the 5 and 3 ends of the linear TFOs were modified with phosphate (PO 4 ) group and hydroxide (OH) group, respectively.", "The designed linear TFOs were synthesized by Dharmacon Research (USA), whereby the 5 and 3 ends of the linear TFOs were modified with phosphate (PO 4 ) group and hydroxide (OH) group, respectively. These modifications were necessary for the circularization of linear TFO. The process of circularization, using the T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs Inc., England), was carried out according to the manufacturer's protocol.", "The process of circularization, using the T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs Inc., England), was carried out according to the manufacturer's protocol. After ligation, the circular TFO RNAs were recovered by ethanol precipitation and the purity of the circular TFO RNAs was measured using spectrophotometer. Denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification.", "Denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. Then, the gel was prerun at 20 to 40 V for 45 minutes.", "Then, the gel was prerun at 20 to 40 V for 45 minutes. Five L of TFO RNA mixed with 5 L of urea loading buffer was heated at 92 ∘ C for 2 minutes and immediately chilled on ice. It was run on the gel at 200 V for 45 minutes.", "It was run on the gel at 200 V for 45 minutes. Finally, the gel was stained with ethidium bromide (Sigma, USA) and viewed with a Bio-Rad Gel Doc XR system (CA, USA). (EMSA) .", "(EMSA) . The target regions of the FIPV genome were synthesized by Dharmacon Research (USA) ( Table 1) . Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours.", "Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours. The sample was run on 15% native polyacrylamide gel at 80 V, in cool condition. The stained gel was viewed by a Bio-Rad Gel Doc XR system.", "The stained gel was viewed by a Bio-Rad Gel Doc XR system. Regions. The binding strength was measured using a nano Isothermal Titration Calorimeter (ITC) (TA instruments, Newcastle, UK).", "The binding strength was measured using a nano Isothermal Titration Calorimeter (ITC) (TA instruments, Newcastle, UK). The RNA sample mixtures, consisting of circular TFOs (0.0002 mM), were incubated with their respective synthetic target regions (0.015 mM) using 1X binding buffer as the diluent. The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections.", "The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer. This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection.", "This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. One hundred nM of TFO RNAs was separately transfected into the CRFK cells using a HiPerFect Transfection Reagent (Qiagen, Germany), as per the manufacturer's protocol. The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours.", "The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C (100 L/well). Finally, the viral inoculum was replaced by fresh maintenance media (MEM containing 1% FBS and 1% pen/strep).", "Finally, the viral inoculum was replaced by fresh maintenance media (MEM containing 1% FBS and 1% pen/strep). Virus-infected and uninfected cells were maintained as positive and negative controls, respectively. The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction.", "The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction. Inhibition. Different concentrations of circular TFO1 RNA (25 nM, 50 nM, 100 nM, and 500 nM) were transfected into CRFK cells.", "Different concentrations of circular TFO1 RNA (25 nM, 50 nM, 100 nM, and 500 nM) were transfected into CRFK cells. The plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ C with 5% CO2. The cells were processed as described above.", "The cells were processed as described above. Madin-Darby Canine Kidney (MDCK) cell (ATCC no. CCL-34), at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. Transfection was performed the same as before.", "Transfection was performed the same as before. One hundred nM of circular TFO RNA was transfected into MDCK cells. Following 6 hours \n\nORF1a/1b and 530-541\n\nORF1a/1b and 7399-7411\n\nORF1a/1b and 14048-14061\n\n- * Highlighted in bold indicated the binding region. * * Unrelated circular TFO. [20, 21] , respectively.", "* * Unrelated circular TFO. [20, 21] , respectively. The reverse transcriptase quantitative real-time PCR (RT-qPCR) was performed using a Bio-Rad CFX96 real-time system (BioRad, USA).", "The reverse transcriptase quantitative real-time PCR (RT-qPCR) was performed using a Bio-Rad CFX96 real-time system (BioRad, USA). The reaction was amplified in a final volume of 25 L using a SensiMix SYBR No-ROX One-Step Kit (Bioline, UK), which consisted of 12.5 L 2X SensiMix SYBR No-Rox One-\n\nStep reaction buffer, 10 M forward and reverse primers, 10 units RiboSafe RNase inhibitor, and 5 L template RNA. Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification.", "Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification. Amount of the virus in the samples was quantified based on this standard curve. Analysis. Data statistical analysis was performed using SPSS 18.0. Data were represented as mean ± SE of three independent tests.", "Data were represented as mean ± SE of three independent tests. One-way ANOVA, Tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation.", "genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region (5 UTR), Open Reading Frames (ORFs) 1a and 1b, and 3 untranslated region (3 UTR) ( Table 1 ). The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex.", "The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. Both ends of the duplex TFOs were ligated with a linker sequence or clamps (C-C) to construct circular TFO RNA. Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO.", "Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE. Target Region.", "Target Region. The binding ability was determined using Electrophoretic Mobility Shift Assay (EMSA) [23] . The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region.", "The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region. The binding ability of different TFO RNAs (TFO1 to TFO5) against their target regions was determined by EMSA (Figure 2) . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band.", "TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band. This indicates the possession of triplex binding ability for all circular TFO RNAs, except TFO2. TFO RNA.", "TFO RNA. Study on the interaction and hybridization of TFO towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. As shown in supplementary Figure 1 (Table 3) . The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection.", "The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection. The results showed viral RNA genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with TFO1, TFO2, TFO3, TFO4, TFO5, and TFO7, respectively. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05).", "The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05). The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ( ≥ 0.05) ( Figure 3 ). The morphological changes of the cells were also captured 72 hours after transfection.", "The morphological changes of the cells were also captured 72 hours after transfection. The cells transfected with circular TFO1, TFO3, TFO4, and TFO5 appeared to be in good condition following virus inoculation, while the cells transfected with circular TFO2 and linear TFO3 and TFO4 showed visible cytopathic effect (CPE), the same as virus inoculated cells (supplementary Figure 2) . Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells.", "Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells. Hence, these results illustrated the capacity of circular TFO RNAs (except TFO2) to inhibit FIPV replication. Concentrations on FIPV Replication. Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs.", "Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs. The experimental conditions were identical to that of the previous experiment, except for TFO1 concentrations of 25 nM, 50 nM, 100 nM, and 500 nM. There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1.", "There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1. The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations ( Figure 4 ).", "However, no significant difference was detected in copy numbers from all of these concentrations ( Figure 4 ). The specificity of the TFO towards FIPV was tested, using TFO1 and TFO5, as the proper representatives of TFOs, on influenza A virus H1N1 New Jersey 8/76. The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary Figure 3 ).", "The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary Figure 3 ). Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift.", "EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. Binding of each circular TFO except circular TFO2 to its respective target forms a complex that migrates slower than unbound TFO. G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively.", "G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively. in the replication process [24] . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins [24] .", "Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins [24] . Hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit FIPV replication. Development of antiviral therapies against FIPV using siRNA [25] and viral protease inhibitors [26] Figure 4 : TFO1 dose-response study for inhibiting FIPV replication.", "Development of antiviral therapies against FIPV using siRNA [25] and viral protease inhibitors [26] Figure 4 : TFO1 dose-response study for inhibiting FIPV replication. The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction.", "50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction. Data are averages of 3 independent tests (mean ± SE). * Significantly different from FIPV-infected group. as potential new treatments against FIPV infection.", "* Significantly different from FIPV-infected group. as potential new treatments against FIPV infection. In this study, circular Triple Helix Forming Oligonucleotide (TFO) RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively.", "TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively. TFO3 to TFO5 targeted different regions of the ORF1a/1b on FIPV genome. Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis.", "Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis. All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO (Figure 1 ). The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate.", "The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate. EMSA was used to show the binding capability of each circular TFO towards the target region in the FIPV genome except for TFO2 which showed lack of formation of complex structure upon hybridization ( Figure 2) . The EMSA result also concurred with the antiviral study, where all circular TFOs (except TFO2) were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells (Figure 3 ).", "The EMSA result also concurred with the antiviral study, where all circular TFOs (except TFO2) were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells (Figure 3 ). However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions. Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems.", "Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems. Since, TFO1 shows the lowest value (Table 3) , the antiviral properties of this TFO were evaluated in doseresponse study. As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication.", "As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication. However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours [27, 28] .", "In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours [27, 28] . The above finding probably also explained the reason why no antiviral effect was detected when the transfection of the TFO was performed on virus-infected cells (data not shown). The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] .", "The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] . Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] .", "Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] . However, there are various factors that may limit the activity of TFOs in cells like intracellular degradation of the TFO and limited accessibility of the TFO to the target sites which can prevent triplex formation [31] . These findings may also explain the inability of the designed TFO1 to inhibit further virus replication in dose-response study (Figure 4) .", "These findings may also explain the inability of the designed TFO1 to inhibit further virus replication in dose-response study (Figure 4) . Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] .", "However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] . Recently, McDonagh et al. [25] developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells.", "[25] developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells. The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus [13] and TFO to inhibit the transcription of 1(I) collagen in rat fibroblasts [14] , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] .", "In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] . These studies indicated the flexibility of using TFO-based oligonucleotides as a potential molecular-based therapy. In this study, we demonstrated short circular TFO RNAs between 28 and 34 mers (Table 1) , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome.", "In this study, we demonstrated short circular TFO RNAs between 28 and 34 mers (Table 1) , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome. All designed circular TFOs (except TFO2) showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication.", "The TFOs that formed triplex structures showed antiviral effects towards FIPV replication. The reason why TFO2 failed to show any interaction with the target region or antiviral activity is probably due to the length of TFO2 (i.e., 24 mers), which might be insufficient to a triplex formation upon hybridization (Figure 2 ), be effective enough to suppress viral RNA transcription, and eventually inhibit virus replication. Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out.", "Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. In vitro antiviral study which showed no antiviral property for unrelated TFO (TFO7) and also inability of circular TFO1 and TFO5 to inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity. In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats.", "In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats. However, further studies on TFO specificity, actual mechanism of circular TFO RNA in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future." ]

[ "Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective.", "Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells.", "RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay.", "The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Text: Feline Infectious Peritonitis Virus (FIPV) is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome.", "Text: Feline Infectious Peritonitis Virus (FIPV) is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome. FIPV is grouped as feline coronavirus (FCoV), under the family Coronaviridae. FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus (FECV), a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV [1] .", "FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus (FECV), a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV [1] . The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory.", "Two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory. Internal mutation theory stated that the development of FIP is due to the exposure of cat to variants of FCoV which have been mutated by gaining the ability to replicate within the macrophages [2] , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population [3] . Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces [4] .", "Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces [4] . About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease [4] . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV.", "The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV. Vaccination against FIPV with an attenuated, temperature-sensitive strain of type II FIPV induces low antibody titre in kittens that have not been exposed to FCoV. However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] .", "However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] . In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement [1] . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] .", "Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] . However, those treatments were ineffective. Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection.", "Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection. Triple Helix Forming Oligonucleotide (TFO) is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA [9] . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] .", "Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] . Hence, TFO has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes [11] [12] [13] [14] [15] [16] . The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication.", "The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication. serotype II strain WSU 79-1146 (ATCC no. VR-1777) was grown in CRFK cells. A serial 10-fold dilution of FIPV was prepared from the working stock.", "A serial 10-fold dilution of FIPV was prepared from the working stock. Confluent 96-well plate was inoculated with 100 L of each virus dilution/well. The plate was incubated in a humidified incubator at 37 ∘ C, 5% CO 2 . Cytopathic effects (CPE) development was observed.", "Cytopathic effects (CPE) development was observed. The results were recorded after 72 hours and the virus tissue culture infective dose 50 (TCID 50 ) was calculated using Reed and Muench's method [17] . Oligonucleotide RNA.", "Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides (TFOs) were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 (Accession no: AY994055) [18] . TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed ( Table 1 ).", "TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed ( Table 1 ). The specificity of the TFOs was identified using BLAST search in the NCBI database. The designed linear TFOs were synthesized by Dharmacon Research (USA), whereby the 5 and 3 ends of the linear TFOs were modified with phosphate (PO 4 ) group and hydroxide (OH) group, respectively.", "The designed linear TFOs were synthesized by Dharmacon Research (USA), whereby the 5 and 3 ends of the linear TFOs were modified with phosphate (PO 4 ) group and hydroxide (OH) group, respectively. These modifications were necessary for the circularization of linear TFO. The process of circularization, using the T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs Inc., England), was carried out according to the manufacturer's protocol.", "The process of circularization, using the T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs Inc., England), was carried out according to the manufacturer's protocol. After ligation, the circular TFO RNAs were recovered by ethanol precipitation and the purity of the circular TFO RNAs was measured using spectrophotometer. Denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification.", "Denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. Then, the gel was prerun at 20 to 40 V for 45 minutes.", "Then, the gel was prerun at 20 to 40 V for 45 minutes. Five L of TFO RNA mixed with 5 L of urea loading buffer was heated at 92 ∘ C for 2 minutes and immediately chilled on ice. It was run on the gel at 200 V for 45 minutes.", "It was run on the gel at 200 V for 45 minutes. Finally, the gel was stained with ethidium bromide (Sigma, USA) and viewed with a Bio-Rad Gel Doc XR system (CA, USA). (EMSA) .", "(EMSA) . The target regions of the FIPV genome were synthesized by Dharmacon Research (USA) ( Table 1) . Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours.", "Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours. The sample was run on 15% native polyacrylamide gel at 80 V, in cool condition. The stained gel was viewed by a Bio-Rad Gel Doc XR system.", "The stained gel was viewed by a Bio-Rad Gel Doc XR system. Regions. The binding strength was measured using a nano Isothermal Titration Calorimeter (ITC) (TA instruments, Newcastle, UK).", "The binding strength was measured using a nano Isothermal Titration Calorimeter (ITC) (TA instruments, Newcastle, UK). The RNA sample mixtures, consisting of circular TFOs (0.0002 mM), were incubated with their respective synthetic target regions (0.015 mM) using 1X binding buffer as the diluent. The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections.", "The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer. This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection.", "This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. One hundred nM of TFO RNAs was separately transfected into the CRFK cells using a HiPerFect Transfection Reagent (Qiagen, Germany), as per the manufacturer's protocol. The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours.", "The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C (100 L/well). Finally, the viral inoculum was replaced by fresh maintenance media (MEM containing 1% FBS and 1% pen/strep).", "Finally, the viral inoculum was replaced by fresh maintenance media (MEM containing 1% FBS and 1% pen/strep). Virus-infected and uninfected cells were maintained as positive and negative controls, respectively. The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction.", "The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction. Inhibition. Different concentrations of circular TFO1 RNA (25 nM, 50 nM, 100 nM, and 500 nM) were transfected into CRFK cells.", "Different concentrations of circular TFO1 RNA (25 nM, 50 nM, 100 nM, and 500 nM) were transfected into CRFK cells. The plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ C with 5% CO2. The cells were processed as described above.", "The cells were processed as described above. Madin-Darby Canine Kidney (MDCK) cell (ATCC no. CCL-34), at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. Transfection was performed the same as before.", "Transfection was performed the same as before. One hundred nM of circular TFO RNA was transfected into MDCK cells. Following 6 hours \n\nORF1a/1b and 530-541\n\nORF1a/1b and 7399-7411\n\nORF1a/1b and 14048-14061\n\n- * Highlighted in bold indicated the binding region. * * Unrelated circular TFO. [20, 21] , respectively.", "* * Unrelated circular TFO. [20, 21] , respectively. The reverse transcriptase quantitative real-time PCR (RT-qPCR) was performed using a Bio-Rad CFX96 real-time system (BioRad, USA).", "The reverse transcriptase quantitative real-time PCR (RT-qPCR) was performed using a Bio-Rad CFX96 real-time system (BioRad, USA). The reaction was amplified in a final volume of 25 L using a SensiMix SYBR No-ROX One-Step Kit (Bioline, UK), which consisted of 12.5 L 2X SensiMix SYBR No-Rox One-\n\nStep reaction buffer, 10 M forward and reverse primers, 10 units RiboSafe RNase inhibitor, and 5 L template RNA. Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification.", "Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification. Amount of the virus in the samples was quantified based on this standard curve. Analysis. Data statistical analysis was performed using SPSS 18.0. Data were represented as mean ± SE of three independent tests.", "Data were represented as mean ± SE of three independent tests. One-way ANOVA, Tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation.", "genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region (5 UTR), Open Reading Frames (ORFs) 1a and 1b, and 3 untranslated region (3 UTR) ( Table 1 ). The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex.", "The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. Both ends of the duplex TFOs were ligated with a linker sequence or clamps (C-C) to construct circular TFO RNA. Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO.", "Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE. Target Region.", "Target Region. The binding ability was determined using Electrophoretic Mobility Shift Assay (EMSA) [23] . The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region.", "The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region. The binding ability of different TFO RNAs (TFO1 to TFO5) against their target regions was determined by EMSA (Figure 2) . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band.", "TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band. This indicates the possession of triplex binding ability for all circular TFO RNAs, except TFO2. TFO RNA.", "TFO RNA. Study on the interaction and hybridization of TFO towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. As shown in supplementary Figure 1 (Table 3) . The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection.", "The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection. The results showed viral RNA genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with TFO1, TFO2, TFO3, TFO4, TFO5, and TFO7, respectively. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05).", "The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05). The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ( ≥ 0.05) ( Figure 3 ). The morphological changes of the cells were also captured 72 hours after transfection.", "The morphological changes of the cells were also captured 72 hours after transfection. The cells transfected with circular TFO1, TFO3, TFO4, and TFO5 appeared to be in good condition following virus inoculation, while the cells transfected with circular TFO2 and linear TFO3 and TFO4 showed visible cytopathic effect (CPE), the same as virus inoculated cells (supplementary Figure 2) . Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells.", "Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells. Hence, these results illustrated the capacity of circular TFO RNAs (except TFO2) to inhibit FIPV replication. Concentrations on FIPV Replication. Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs.", "Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs. The experimental conditions were identical to that of the previous experiment, except for TFO1 concentrations of 25 nM, 50 nM, 100 nM, and 500 nM. There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1.", "There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1. The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations ( Figure 4 ).", "However, no significant difference was detected in copy numbers from all of these concentrations ( Figure 4 ). The specificity of the TFO towards FIPV was tested, using TFO1 and TFO5, as the proper representatives of TFOs, on influenza A virus H1N1 New Jersey 8/76. The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary Figure 3 ).", "The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary Figure 3 ). Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift.", "EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. Binding of each circular TFO except circular TFO2 to its respective target forms a complex that migrates slower than unbound TFO. G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively.", "G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively. in the replication process [24] . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins [24] .", "Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins [24] . Hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit FIPV replication. Development of antiviral therapies against FIPV using siRNA [25] and viral protease inhibitors [26] Figure 4 : TFO1 dose-response study for inhibiting FIPV replication.", "Development of antiviral therapies against FIPV using siRNA [25] and viral protease inhibitors [26] Figure 4 : TFO1 dose-response study for inhibiting FIPV replication. The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction.", "50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction. Data are averages of 3 independent tests (mean ± SE). * Significantly different from FIPV-infected group. as potential new treatments against FIPV infection.", "* Significantly different from FIPV-infected group. as potential new treatments against FIPV infection. In this study, circular Triple Helix Forming Oligonucleotide (TFO) RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively.", "TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively. TFO3 to TFO5 targeted different regions of the ORF1a/1b on FIPV genome. Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis.", "Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis. All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO (Figure 1 ). The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate.", "The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate. EMSA was used to show the binding capability of each circular TFO towards the target region in the FIPV genome except for TFO2 which showed lack of formation of complex structure upon hybridization ( Figure 2) . The EMSA result also concurred with the antiviral study, where all circular TFOs (except TFO2) were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells (Figure 3 ).", "The EMSA result also concurred with the antiviral study, where all circular TFOs (except TFO2) were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells (Figure 3 ). However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions. Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems.", "Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems. Since, TFO1 shows the lowest value (Table 3) , the antiviral properties of this TFO were evaluated in doseresponse study. As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication.", "As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication. However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours [27, 28] .", "In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours [27, 28] . The above finding probably also explained the reason why no antiviral effect was detected when the transfection of the TFO was performed on virus-infected cells (data not shown). The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] .", "The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] . Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] .", "Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] . However, there are various factors that may limit the activity of TFOs in cells like intracellular degradation of the TFO and limited accessibility of the TFO to the target sites which can prevent triplex formation [31] . These findings may also explain the inability of the designed TFO1 to inhibit further virus replication in dose-response study (Figure 4) .", "These findings may also explain the inability of the designed TFO1 to inhibit further virus replication in dose-response study (Figure 4) . Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] .", "However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] . Recently, McDonagh et al. [25] developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells.", "[25] developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells. The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus [13] and TFO to inhibit the transcription of 1(I) collagen in rat fibroblasts [14] , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] .", "In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] . These studies indicated the flexibility of using TFO-based oligonucleotides as a potential molecular-based therapy. In this study, we demonstrated short circular TFO RNAs between 28 and 34 mers (Table 1) , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome.", "In this study, we demonstrated short circular TFO RNAs between 28 and 34 mers (Table 1) , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome. All designed circular TFOs (except TFO2) showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication.", "The TFOs that formed triplex structures showed antiviral effects towards FIPV replication. The reason why TFO2 failed to show any interaction with the target region or antiviral activity is probably due to the length of TFO2 (i.e., 24 mers), which might be insufficient to a triplex formation upon hybridization (Figure 2 ), be effective enough to suppress viral RNA transcription, and eventually inhibit virus replication. Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out.", "Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. In vitro antiviral study which showed no antiviral property for unrelated TFO (TFO7) and also inability of circular TFO1 and TFO5 to inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity. In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats.", "In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats. However, further studies on TFO specificity, actual mechanism of circular TFO RNA in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future." ]

[ "Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective.", "Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells.", "RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay.", "The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Text: Feline Infectious Peritonitis Virus (FIPV) is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome.", "Text: Feline Infectious Peritonitis Virus (FIPV) is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome. FIPV is grouped as feline coronavirus (FCoV), under the family Coronaviridae. FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus (FECV), a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV [1] .", "FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus (FECV), a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV [1] . The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory.", "Two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory. Internal mutation theory stated that the development of FIP is due to the exposure of cat to variants of FCoV which have been mutated by gaining the ability to replicate within the macrophages [2] , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population [3] . Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces [4] .", "Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces [4] . About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease [4] . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV.", "The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV. Vaccination against FIPV with an attenuated, temperature-sensitive strain of type II FIPV induces low antibody titre in kittens that have not been exposed to FCoV. However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] .", "However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] . In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement [1] . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] .", "Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] . However, those treatments were ineffective. Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection.", "Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection. Triple Helix Forming Oligonucleotide (TFO) is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA [9] . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] .", "Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] . Hence, TFO has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes [11] [12] [13] [14] [15] [16] . The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication.", "The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication. serotype II strain WSU 79-1146 (ATCC no. VR-1777) was grown in CRFK cells. A serial 10-fold dilution of FIPV was prepared from the working stock.", "A serial 10-fold dilution of FIPV was prepared from the working stock. Confluent 96-well plate was inoculated with 100 L of each virus dilution/well. The plate was incubated in a humidified incubator at 37 ∘ C, 5% CO 2 . Cytopathic effects (CPE) development was observed.", "Cytopathic effects (CPE) development was observed. The results were recorded after 72 hours and the virus tissue culture infective dose 50 (TCID 50 ) was calculated using Reed and Muench's method [17] . Oligonucleotide RNA.", "Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides (TFOs) were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 (Accession no: AY994055) [18] . TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed ( Table 1 ).", "TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed ( Table 1 ). The specificity of the TFOs was identified using BLAST search in the NCBI database. The designed linear TFOs were synthesized by Dharmacon Research (USA), whereby the 5 and 3 ends of the linear TFOs were modified with phosphate (PO 4 ) group and hydroxide (OH) group, respectively.", "The designed linear TFOs were synthesized by Dharmacon Research (USA), whereby the 5 and 3 ends of the linear TFOs were modified with phosphate (PO 4 ) group and hydroxide (OH) group, respectively. These modifications were necessary for the circularization of linear TFO. The process of circularization, using the T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs Inc., England), was carried out according to the manufacturer's protocol.", "The process of circularization, using the T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs Inc., England), was carried out according to the manufacturer's protocol. After ligation, the circular TFO RNAs were recovered by ethanol precipitation and the purity of the circular TFO RNAs was measured using spectrophotometer. Denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification.", "Denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. Then, the gel was prerun at 20 to 40 V for 45 minutes.", "Then, the gel was prerun at 20 to 40 V for 45 minutes. Five L of TFO RNA mixed with 5 L of urea loading buffer was heated at 92 ∘ C for 2 minutes and immediately chilled on ice. It was run on the gel at 200 V for 45 minutes.", "It was run on the gel at 200 V for 45 minutes. Finally, the gel was stained with ethidium bromide (Sigma, USA) and viewed with a Bio-Rad Gel Doc XR system (CA, USA). (EMSA) .", "(EMSA) . The target regions of the FIPV genome were synthesized by Dharmacon Research (USA) ( Table 1) . Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours.", "Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours. The sample was run on 15% native polyacrylamide gel at 80 V, in cool condition. The stained gel was viewed by a Bio-Rad Gel Doc XR system.", "The stained gel was viewed by a Bio-Rad Gel Doc XR system. Regions. The binding strength was measured using a nano Isothermal Titration Calorimeter (ITC) (TA instruments, Newcastle, UK).", "The binding strength was measured using a nano Isothermal Titration Calorimeter (ITC) (TA instruments, Newcastle, UK). The RNA sample mixtures, consisting of circular TFOs (0.0002 mM), were incubated with their respective synthetic target regions (0.015 mM) using 1X binding buffer as the diluent. The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections.", "The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer. This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection.", "This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. One hundred nM of TFO RNAs was separately transfected into the CRFK cells using a HiPerFect Transfection Reagent (Qiagen, Germany), as per the manufacturer's protocol. The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours.", "The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C (100 L/well). Finally, the viral inoculum was replaced by fresh maintenance media (MEM containing 1% FBS and 1% pen/strep).", "Finally, the viral inoculum was replaced by fresh maintenance media (MEM containing 1% FBS and 1% pen/strep). Virus-infected and uninfected cells were maintained as positive and negative controls, respectively. The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction.", "The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction. Inhibition. Different concentrations of circular TFO1 RNA (25 nM, 50 nM, 100 nM, and 500 nM) were transfected into CRFK cells.", "Different concentrations of circular TFO1 RNA (25 nM, 50 nM, 100 nM, and 500 nM) were transfected into CRFK cells. The plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ C with 5% CO2. The cells were processed as described above.", "The cells were processed as described above. Madin-Darby Canine Kidney (MDCK) cell (ATCC no. CCL-34), at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. Transfection was performed the same as before.", "Transfection was performed the same as before. One hundred nM of circular TFO RNA was transfected into MDCK cells. Following 6 hours \n\nORF1a/1b and 530-541\n\nORF1a/1b and 7399-7411\n\nORF1a/1b and 14048-14061\n\n- * Highlighted in bold indicated the binding region. * * Unrelated circular TFO. [20, 21] , respectively.", "* * Unrelated circular TFO. [20, 21] , respectively. The reverse transcriptase quantitative real-time PCR (RT-qPCR) was performed using a Bio-Rad CFX96 real-time system (BioRad, USA).", "The reverse transcriptase quantitative real-time PCR (RT-qPCR) was performed using a Bio-Rad CFX96 real-time system (BioRad, USA). The reaction was amplified in a final volume of 25 L using a SensiMix SYBR No-ROX One-Step Kit (Bioline, UK), which consisted of 12.5 L 2X SensiMix SYBR No-Rox One-\n\nStep reaction buffer, 10 M forward and reverse primers, 10 units RiboSafe RNase inhibitor, and 5 L template RNA. Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification.", "Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification. Amount of the virus in the samples was quantified based on this standard curve. Analysis. Data statistical analysis was performed using SPSS 18.0. Data were represented as mean ± SE of three independent tests.", "Data were represented as mean ± SE of three independent tests. One-way ANOVA, Tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation.", "genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region (5 UTR), Open Reading Frames (ORFs) 1a and 1b, and 3 untranslated region (3 UTR) ( Table 1 ). The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex.", "The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. Both ends of the duplex TFOs were ligated with a linker sequence or clamps (C-C) to construct circular TFO RNA. Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO.", "Denaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE. Target Region.", "Target Region. The binding ability was determined using Electrophoretic Mobility Shift Assay (EMSA) [23] . The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region.", "The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region. The binding ability of different TFO RNAs (TFO1 to TFO5) against their target regions was determined by EMSA (Figure 2) . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band.", "TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band. This indicates the possession of triplex binding ability for all circular TFO RNAs, except TFO2. TFO RNA.", "TFO RNA. Study on the interaction and hybridization of TFO towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. As shown in supplementary Figure 1 (Table 3) . The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection.", "The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection. The results showed viral RNA genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with TFO1, TFO2, TFO3, TFO4, TFO5, and TFO7, respectively. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05).", "The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05). The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ( ≥ 0.05) ( Figure 3 ). The morphological changes of the cells were also captured 72 hours after transfection.", "The morphological changes of the cells were also captured 72 hours after transfection. The cells transfected with circular TFO1, TFO3, TFO4, and TFO5 appeared to be in good condition following virus inoculation, while the cells transfected with circular TFO2 and linear TFO3 and TFO4 showed visible cytopathic effect (CPE), the same as virus inoculated cells (supplementary Figure 2) . Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells.", "Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells. Hence, these results illustrated the capacity of circular TFO RNAs (except TFO2) to inhibit FIPV replication. Concentrations on FIPV Replication. Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs.", "Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs. The experimental conditions were identical to that of the previous experiment, except for TFO1 concentrations of 25 nM, 50 nM, 100 nM, and 500 nM. There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1.", "There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1. The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations ( Figure 4 ).", "However, no significant difference was detected in copy numbers from all of these concentrations ( Figure 4 ). The specificity of the TFO towards FIPV was tested, using TFO1 and TFO5, as the proper representatives of TFOs, on influenza A virus H1N1 New Jersey 8/76. The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary Figure 3 ).", "The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary Figure 3 ). Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift.", "EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. Binding of each circular TFO except circular TFO2 to its respective target forms a complex that migrates slower than unbound TFO. G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively.", "G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively. in the replication process [24] . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins [24] .", "Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins [24] . Hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit FIPV replication. Development of antiviral therapies against FIPV using siRNA [25] and viral protease inhibitors [26] Figure 4 : TFO1 dose-response study for inhibiting FIPV replication.", "Development of antiviral therapies against FIPV using siRNA [25] and viral protease inhibitors [26] Figure 4 : TFO1 dose-response study for inhibiting FIPV replication. The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction.", "50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction. Data are averages of 3 independent tests (mean ± SE). * Significantly different from FIPV-infected group. as potential new treatments against FIPV infection.", "* Significantly different from FIPV-infected group. as potential new treatments against FIPV infection. In this study, circular Triple Helix Forming Oligonucleotide (TFO) RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively.", "TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively. TFO3 to TFO5 targeted different regions of the ORF1a/1b on FIPV genome. Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis.", "Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis. All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO (Figure 1 ). The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate.", "The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate. EMSA was used to show the binding capability of each circular TFO towards the target region in the FIPV genome except for TFO2 which showed lack of formation of complex structure upon hybridization ( Figure 2) . The EMSA result also concurred with the antiviral study, where all circular TFOs (except TFO2) were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells (Figure 3 ).", "The EMSA result also concurred with the antiviral study, where all circular TFOs (except TFO2) were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells (Figure 3 ). However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions. Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems.", "Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems. Since, TFO1 shows the lowest value (Table 3) , the antiviral properties of this TFO were evaluated in doseresponse study. As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication.", "As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication. However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours [27, 28] .", "In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours [27, 28] . The above finding probably also explained the reason why no antiviral effect was detected when the transfection of the TFO was performed on virus-infected cells (data not shown). The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] .", "The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] . Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] .", "Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] . However, there are various factors that may limit the activity of TFOs in cells like intracellular degradation of the TFO and limited accessibility of the TFO to the target sites which can prevent triplex formation [31] . These findings may also explain the inability of the designed TFO1 to inhibit further virus replication in dose-response study (Figure 4) .", "These findings may also explain the inability of the designed TFO1 to inhibit further virus replication in dose-response study (Figure 4) . Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] .", "However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] . Recently, McDonagh et al. [25] developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells.", "[25] developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells. The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus [13] and TFO to inhibit the transcription of 1(I) collagen in rat fibroblasts [14] , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] .", "In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] . These studies indicated the flexibility of using TFO-based oligonucleotides as a potential molecular-based therapy. In this study, we demonstrated short circular TFO RNAs between 28 and 34 mers (Table 1) , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome.", "In this study, we demonstrated short circular TFO RNAs between 28 and 34 mers (Table 1) , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome. All designed circular TFOs (except TFO2) showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication.", "The TFOs that formed triplex structures showed antiviral effects towards FIPV replication. The reason why TFO2 failed to show any interaction with the target region or antiviral activity is probably due to the length of TFO2 (i.e., 24 mers), which might be insufficient to a triplex formation upon hybridization (Figure 2 ), be effective enough to suppress viral RNA transcription, and eventually inhibit virus replication. Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out.", "Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. In vitro antiviral study which showed no antiviral property for unrelated TFO (TFO7) and also inability of circular TFO1 and TFO5 to inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity. In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats.", "In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats. However, further studies on TFO specificity, actual mechanism of circular TFO RNA in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future." ]

[ "In the present study, the anti-influenza A (H2N2) virus activity of patchouli alcohol was studied in vitro, in vivo and in silico. The CC(50) of patchouli alcohol was above 20 µM. Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM.", "Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM. MTT assay showed that the inhibition by patchouli alcohol appears strongly after penetration of the virus into the cell. In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day.", "In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day. Flexible docking and molecular dynamic simulations indicated that patchouli alcohol was bound to the neuraminidase protein of influenza virus, with an interaction energy of –40.38 kcal mol(–1). The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process.", "The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process. Based on spatial and energetic criteria, patchouli alcohol interfered with the NA functions. Results presented here suggest that patchouli alcohol possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry.", "Results presented here suggest that patchouli alcohol possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry. Text: The influenza virus, which is one of the main causes of acute respiratory infections in humans, can lead to annual epidemics and infrequent pandemics. The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] .", "The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] . Today, their descendants continue to cause the majority of influenza infections in humans [3] . So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] .", "So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] . The release of new virions from the infected cell is a key step in the influenza life cycle and need neuraminidase (NA) to cleave the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue [6] . The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] .", "The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] . Consistent efforts have been devoted to the development of NA inhibitors, using the crystal structure of the N2 sub-type NA protein [8] [9] [10] [11] [12] [13] [14] [15] . Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] .", "Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] . However, with an increase in medical use, the oseltamivir-resistant strains have been found and probably lead to a large scale outbreak of novel pandemic flu [16, 17] . Patchouli alcohol ( Figure 1 ) has been well known for over a century.", "Patchouli alcohol ( Figure 1 ) has been well known for over a century. It is a major constituent of the pungent oil from the East Indian shrub Pogostemon cablin (Blanco) Benth, and widely used in fragrances. Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] .", "Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] . The essential oil is very appreciated for its characteristic pleasant and long lasting woody, earthy, and camphoraceous odor, as well as for its fixative properties, being suitable for use in soaps and cosmetic products [16, 17] . The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] .", "The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] . Moreover, the plant is widely used in Traditional Chinese Medicine as it presents various types of pharmacological activity according to the composition of the oil [16, 17] . Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] .", "Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] . To the best of our knowledge, the anti-influenza virus (H2N2) activities of patchouli alcohol have not been evaluated yet. Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model.", "Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model. On such basis, explicitly solvated docking and molecular dynamic (MD) methods were applied to investigative the binding mode involving patchouli alcohol with influenza virus NA protein. We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs.", "We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs. First the efficacy of patchouli alcohol on influenza A (H2N2) virus replication and cell viability were examined. CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK.", "CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK. The CC 50 of patchouli alcohol was above 20 mM, which indicated that patchouli alcohol did not affect the growth of MDCK (Table 1) . Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity.", "Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity. Moreover, patchouli alcohol was found to inhibit influenza A (H2N2) virus with an IC 50 of 4.03 ± 0.23 µM. Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96.", "Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96. It is reported that a SI of 4 or more is appropriate for an antiviral agent [18] , suggesting that patchouli alcohol can be judged to have anti-influenza A (H2N2) virus activity. Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM.", "Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM. Furthermore, it showed weak activity against B/Ibaraki/2/85 (IC 50 = 40.82 µM) [19] . With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol.", "With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol. Cells were pretreated with patchouli alcohol prior to virus infection (pretreatment cells), viruses were pretreated prior to infection (pretreatment virus), and patchouli alcohol was added during the adsorption period (adsorption) or after penetration of the viruses into cells (replication). Experiments were repeated independently three times and data presented are the average of three experiments.", "Experiments were repeated independently three times and data presented are the average of three experiments. The symbols * indicated very significant difference p < 0.01 with respect to other mode (pretreatment virus, adsorption and pretreatment cell). As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner.", "As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner. It showed best antiviral activity when added at a concentration of 8 µM during the replication period with inhibition of the viral replication of 97.68% ± 2.09% for influenza A (H2N2) at 72 h. However, no significant effect was detected when patchouli alcohol was used for pretreatment of cells or viruses or when patchouli alcohol was only added during the adsorption phase. These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell.", "These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell. Besides, biochemical studies have indicated that the bioactivity of NA protein is essential determinant after the replication of influenza A (H2N2) virus [20] [21] [22] . Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol.", "Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol. To evaluate the toxicity of patchouli alcohol, the mean value of body weight of mice in each group was statistically analyzed. The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05).", "The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05). Physiological status was observed in virus infection mice. Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc.", "Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc. In the mouse influenza model, viral infection leads to loss of body weight and high mortality. Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative to untreated infected (control) animals.", "Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative to untreated infected (control) animals. A comparison of efficacy of patchouli alcohol and oseltamivir in vivo mouse influenza model (oral treatment) showed that at a dose of 5 mg/kg/day, patchouli alcohol showed obvious protection against the influenza virus, as the mean day to death was detected as 11.8 ± 1.1 (Table 2) . When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8.", "When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8. Whereas oseltamivir at this dose level (1 mg/kg/day) showed 50% protection (measured by survivors/total) against the influenza virus. In the H2N2 infected control group, there were no survivors.", "In the H2N2 infected control group, there were no survivors. In view of both in vitro and in vivo data, we conclude that patchouli alcohol could be used in the treatment of human influenza virus infections. Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein.", "Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein. As the total energies and backbone root-mean-square-deviations (RMSD) in Figure 3 indicate, the energy-minimized patchouli alcohol-NA complex has been in equilibrium since about 0.5 ns, and then retains quite stable in the last 19.5 ns. It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] .", "It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] . Accordingly, the geometric and energetic analyses were made on the average structures of 0.5~20.0 ns MD trajectories, where the system has been already at equilibrium. The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ).", "The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ). As shown in Figure 4 , the patchouli alcohol was bound at the active site which also bound to oseltamivir and zanamivir [28] . As Figure 5 shows, the oxygen atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue.", "As Figure 5 shows, the oxygen atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue. The values of distances in Figure 6 further reveal that the docked complex remains rather stable throughout the simulation, with the average distances of Glu119:OE2patchouli alcohol:O and Tyr406:OH -patchouli alcohol:O less than 2.8 Å. The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) .", "The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) . Besides, patchouli alcohol was stabilized by residues Arg118, Asp151, Arg152, Trp178, Ala246, Glu276, Arg292, Asn294 and Gln347, especially residues Asp151, Arg152 and Glu276 ( Figure 5 and Table 3 ). As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] .", "As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] . The catalytic residues Asp151, Arg152 and Glu276 are crucial to the NA functions and the residues Glu119 and Tyr406 are important to stabilize the NA active sites [32, 33] . It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments.", "It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments. Patchouli alcohol matches with the NA active site and has an acceptable interaction energy. Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents.", "Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents. Patchouli alcohol and oseltamivir were obtained from Sigma Chemical Co. (St. Louis, MO, USA, purity > 99%) and was stored in glass vials with Teflon sealed caps at −20 ± 0.5 °C in the absence of light. MDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China).", "MDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China). The cells were grown in monolayer culture with Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin. The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent.", "The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent. Cells were plated out onto 96-well culture plates for cytotoxicity and anti-influenza assays, and propagated at 37 °C in an atmosphere of 5% CO 2 . The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China).", "The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China). Virus was routinely grown on MDCK cells. The stock cultures were prepared from supernatants of infected cells and stored at −80 °C. The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method.", "The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method. Briefly, cells were seeded on a microtiter plate in the absence or presence of various concentrations (20 µM -0.0098 µM) of patchouli alcohol (eight replicates) and incubated at 37 °C in a humidified atmosphere of 5% CO 2 for 72 h. The supernatants were discarded, washed with PBS twice and MTT reagent (5 mg/mL in PBS) was added to each well. After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min.", "After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min. After that the plates were read on an ELISA reader (Thermo Molecular Devices Co., Union City, USA) at 570/630 nm. The mean OD of the cell control wells was assigned a value of 100%.", "The mean OD of the cell control wells was assigned a value of 100%. The maximal non-toxic concentration (TD 0 ) and 50% cytotoxic concentration (CC 50 ) were calculated by linear regression analysis of the dose-response curves generated from the data. Inhibition of virus replication was measured by the MTT method.", "Inhibition of virus replication was measured by the MTT method. Serial dilution of the treated virus was adsorbed to the cells for 1 h at 37 °C. The residual inoculum was discared and infected cells were added with EMEM containing 2% FCS. Each assay was performed in eight replicates.", "Each assay was performed in eight replicates. After incubation for 72 h at 37 °C, the cultures were measured by MTT method as described above. The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves.", "The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves. Cells and viruses were incubated with patchouli alcohol at different stages during the viral infection cycle in order to determine the mode of antiviral action. Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells.", "Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells. Patchouli alcohol was always used at the nontoxic concentration. Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C.", "Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C. The compound was aspirated and cells were washed immediately before the influenza A (H2N2) inoculum was added. For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells.", "For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells. For analyzing the anti-influenza A (H2N2) inhibition during the adsorption period, the same amount of influenza A (H2N2) was mixed with the drug and added to the cells immediately. After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells.", "After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells. The effect of patchouli alcohol against influenza A (H2N2) was also tested during the replication period by adding it after adsorption, as typical performed in anti-influenza A (H2N2) susceptibility studies. Each assay was run in eight replicates.", "Each assay was run in eight replicates. Kunming mice, weighing 18-22 g (6 weeks of age) were purchased from Harbin Veterinary Research Institute Animal Co., Ltd. (Harbin, Heilongjiang, China) . First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline).", "First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline). The mice were orally administered with 10 mg/kg/dose patchouli alcohol, 2 mg/kg/dose patchouli alcohol or 2 mg/kg/dose oseltamivir (dissolved in 2% DMSO in physiological saline) one time daily for 7 days. The weight of mice was determined daily.", "The weight of mice was determined daily. We conducted procedures according to Principle of Laboratory Animal Care (NIH Publication No. 85 -23, revised 1985) and the guidelines of the Peking University Animal Research Committee. Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation.", "Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation. Drugs were prepared in 2% DMSO in physiological saline and administered 4 h prior to virus exposure and continued daily for 5 days. All mice were observed daily for changes in weight and for any deaths.", "All mice were observed daily for changes in weight and for any deaths. Parameters for evaluation of antiviral activity included weight loss, reduction in mortality and/or increase in mean day to death (MDD) determined through 15 days. The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] .", "The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] . For convenience, the structure is named as NA hereafter. Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 .", "Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 . The docking and molecular dynamics (MD) simulations were performed by the general protocols in the Insight II 2005 software packages, consistent with the previous literatures [24, 26, 28, 35, [37] [38] [39] . During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K).", "During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K). The MD temperature was controlled by the velocity scaling thermostat [40] . Integrations of the classical equations of motion were achieved using the Verlet algorithm.", "Integrations of the classical equations of motion were achieved using the Verlet algorithm. The systems were solvated in a large sphere of TIP3P water molecules [40] with the radius of 35.0 Å, which is enough to hold the ensembles [40] . The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals.", "The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals. The interaction energies of patchouli alcohol with NA and the respective residues at the NA active site were calculated by the Docking module [35], over the 0.5~20.0 ns MD trajectories. All results are expressed as mean values ± standard deviations (SDs) (n = 3).", "All results are expressed as mean values ± standard deviations (SDs) (n = 3). The significance of difference was calculated by one-way analysis of variance, and values p < 0.001 were considered to be significant. In conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate.", "In conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate. Our results provide the promising information for the potential use of patchouli alcohol in the treatment of influenza A (H2N2) virus infectious disease. Further mechanistic studies on the anti-influenza A virus activity are needed to support this point of view." ]

[ "In the present study, the anti-influenza A (H2N2) virus activity of patchouli alcohol was studied in vitro, in vivo and in silico. The CC(50) of patchouli alcohol was above 20 µM. Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM.", "Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM. MTT assay showed that the inhibition by patchouli alcohol appears strongly after penetration of the virus into the cell. In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day.", "In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day. Flexible docking and molecular dynamic simulations indicated that patchouli alcohol was bound to the neuraminidase protein of influenza virus, with an interaction energy of –40.38 kcal mol(–1). The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process.", "The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process. Based on spatial and energetic criteria, patchouli alcohol interfered with the NA functions. Results presented here suggest that patchouli alcohol possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry.", "Results presented here suggest that patchouli alcohol possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry. Text: The influenza virus, which is one of the main causes of acute respiratory infections in humans, can lead to annual epidemics and infrequent pandemics. The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] .", "The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] . Today, their descendants continue to cause the majority of influenza infections in humans [3] . So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] .", "So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] . The release of new virions from the infected cell is a key step in the influenza life cycle and need neuraminidase (NA) to cleave the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue [6] . The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] .", "The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] . Consistent efforts have been devoted to the development of NA inhibitors, using the crystal structure of the N2 sub-type NA protein [8] [9] [10] [11] [12] [13] [14] [15] . Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] .", "Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] . However, with an increase in medical use, the oseltamivir-resistant strains have been found and probably lead to a large scale outbreak of novel pandemic flu [16, 17] . Patchouli alcohol ( Figure 1 ) has been well known for over a century.", "Patchouli alcohol ( Figure 1 ) has been well known for over a century. It is a major constituent of the pungent oil from the East Indian shrub Pogostemon cablin (Blanco) Benth, and widely used in fragrances. Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] .", "Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] . The essential oil is very appreciated for its characteristic pleasant and long lasting woody, earthy, and camphoraceous odor, as well as for its fixative properties, being suitable for use in soaps and cosmetic products [16, 17] . The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] .", "The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] . Moreover, the plant is widely used in Traditional Chinese Medicine as it presents various types of pharmacological activity according to the composition of the oil [16, 17] . Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] .", "Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] . To the best of our knowledge, the anti-influenza virus (H2N2) activities of patchouli alcohol have not been evaluated yet. Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model.", "Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model. On such basis, explicitly solvated docking and molecular dynamic (MD) methods were applied to investigative the binding mode involving patchouli alcohol with influenza virus NA protein. We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs.", "We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs. First the efficacy of patchouli alcohol on influenza A (H2N2) virus replication and cell viability were examined. CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK.", "CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK. The CC 50 of patchouli alcohol was above 20 mM, which indicated that patchouli alcohol did not affect the growth of MDCK (Table 1) . Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity.", "Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity. Moreover, patchouli alcohol was found to inhibit influenza A (H2N2) virus with an IC 50 of 4.03 ± 0.23 µM. Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96.", "Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96. It is reported that a SI of 4 or more is appropriate for an antiviral agent [18] , suggesting that patchouli alcohol can be judged to have anti-influenza A (H2N2) virus activity. Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM.", "Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM. Furthermore, it showed weak activity against B/Ibaraki/2/85 (IC 50 = 40.82 µM) [19] . With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol.", "With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol. Cells were pretreated with patchouli alcohol prior to virus infection (pretreatment cells), viruses were pretreated prior to infection (pretreatment virus), and patchouli alcohol was added during the adsorption period (adsorption) or after penetration of the viruses into cells (replication). Experiments were repeated independently three times and data presented are the average of three experiments.", "Experiments were repeated independently three times and data presented are the average of three experiments. The symbols * indicated very significant difference p < 0.01 with respect to other mode (pretreatment virus, adsorption and pretreatment cell). As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner.", "As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner. It showed best antiviral activity when added at a concentration of 8 µM during the replication period with inhibition of the viral replication of 97.68% ± 2.09% for influenza A (H2N2) at 72 h. However, no significant effect was detected when patchouli alcohol was used for pretreatment of cells or viruses or when patchouli alcohol was only added during the adsorption phase. These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell.", "These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell. Besides, biochemical studies have indicated that the bioactivity of NA protein is essential determinant after the replication of influenza A (H2N2) virus [20] [21] [22] . Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol.", "Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol. To evaluate the toxicity of patchouli alcohol, the mean value of body weight of mice in each group was statistically analyzed. The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05).", "The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05). Physiological status was observed in virus infection mice. Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc.", "Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc. In the mouse influenza model, viral infection leads to loss of body weight and high mortality. Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative to untreated infected (control) animals.", "Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative to untreated infected (control) animals. A comparison of efficacy of patchouli alcohol and oseltamivir in vivo mouse influenza model (oral treatment) showed that at a dose of 5 mg/kg/day, patchouli alcohol showed obvious protection against the influenza virus, as the mean day to death was detected as 11.8 ± 1.1 (Table 2) . When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8.", "When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8. Whereas oseltamivir at this dose level (1 mg/kg/day) showed 50% protection (measured by survivors/total) against the influenza virus. In the H2N2 infected control group, there were no survivors.", "In the H2N2 infected control group, there were no survivors. In view of both in vitro and in vivo data, we conclude that patchouli alcohol could be used in the treatment of human influenza virus infections. Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein.", "Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein. As the total energies and backbone root-mean-square-deviations (RMSD) in Figure 3 indicate, the energy-minimized patchouli alcohol-NA complex has been in equilibrium since about 0.5 ns, and then retains quite stable in the last 19.5 ns. It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] .", "It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] . Accordingly, the geometric and energetic analyses were made on the average structures of 0.5~20.0 ns MD trajectories, where the system has been already at equilibrium. The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ).", "The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ). As shown in Figure 4 , the patchouli alcohol was bound at the active site which also bound to oseltamivir and zanamivir [28] . As Figure 5 shows, the oxygen atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue.", "As Figure 5 shows, the oxygen atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue. The values of distances in Figure 6 further reveal that the docked complex remains rather stable throughout the simulation, with the average distances of Glu119:OE2patchouli alcohol:O and Tyr406:OH -patchouli alcohol:O less than 2.8 Å. The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) .", "The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) . Besides, patchouli alcohol was stabilized by residues Arg118, Asp151, Arg152, Trp178, Ala246, Glu276, Arg292, Asn294 and Gln347, especially residues Asp151, Arg152 and Glu276 ( Figure 5 and Table 3 ). As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] .", "As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] . The catalytic residues Asp151, Arg152 and Glu276 are crucial to the NA functions and the residues Glu119 and Tyr406 are important to stabilize the NA active sites [32, 33] . It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments.", "It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments. Patchouli alcohol matches with the NA active site and has an acceptable interaction energy. Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents.", "Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents. Patchouli alcohol and oseltamivir were obtained from Sigma Chemical Co. (St. Louis, MO, USA, purity > 99%) and was stored in glass vials with Teflon sealed caps at −20 ± 0.5 °C in the absence of light. MDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China).", "MDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China). The cells were grown in monolayer culture with Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin. The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent.", "The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent. Cells were plated out onto 96-well culture plates for cytotoxicity and anti-influenza assays, and propagated at 37 °C in an atmosphere of 5% CO 2 . The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China).", "The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China). Virus was routinely grown on MDCK cells. The stock cultures were prepared from supernatants of infected cells and stored at −80 °C. The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method.", "The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method. Briefly, cells were seeded on a microtiter plate in the absence or presence of various concentrations (20 µM -0.0098 µM) of patchouli alcohol (eight replicates) and incubated at 37 °C in a humidified atmosphere of 5% CO 2 for 72 h. The supernatants were discarded, washed with PBS twice and MTT reagent (5 mg/mL in PBS) was added to each well. After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min.", "After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min. After that the plates were read on an ELISA reader (Thermo Molecular Devices Co., Union City, USA) at 570/630 nm. The mean OD of the cell control wells was assigned a value of 100%.", "The mean OD of the cell control wells was assigned a value of 100%. The maximal non-toxic concentration (TD 0 ) and 50% cytotoxic concentration (CC 50 ) were calculated by linear regression analysis of the dose-response curves generated from the data. Inhibition of virus replication was measured by the MTT method.", "Inhibition of virus replication was measured by the MTT method. Serial dilution of the treated virus was adsorbed to the cells for 1 h at 37 °C. The residual inoculum was discared and infected cells were added with EMEM containing 2% FCS. Each assay was performed in eight replicates.", "Each assay was performed in eight replicates. After incubation for 72 h at 37 °C, the cultures were measured by MTT method as described above. The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves.", "The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves. Cells and viruses were incubated with patchouli alcohol at different stages during the viral infection cycle in order to determine the mode of antiviral action. Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells.", "Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells. Patchouli alcohol was always used at the nontoxic concentration. Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C.", "Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C. The compound was aspirated and cells were washed immediately before the influenza A (H2N2) inoculum was added. For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells.", "For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells. For analyzing the anti-influenza A (H2N2) inhibition during the adsorption period, the same amount of influenza A (H2N2) was mixed with the drug and added to the cells immediately. After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells.", "After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells. The effect of patchouli alcohol against influenza A (H2N2) was also tested during the replication period by adding it after adsorption, as typical performed in anti-influenza A (H2N2) susceptibility studies. Each assay was run in eight replicates.", "Each assay was run in eight replicates. Kunming mice, weighing 18-22 g (6 weeks of age) were purchased from Harbin Veterinary Research Institute Animal Co., Ltd. (Harbin, Heilongjiang, China) . First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline).", "First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline). The mice were orally administered with 10 mg/kg/dose patchouli alcohol, 2 mg/kg/dose patchouli alcohol or 2 mg/kg/dose oseltamivir (dissolved in 2% DMSO in physiological saline) one time daily for 7 days. The weight of mice was determined daily.", "The weight of mice was determined daily. We conducted procedures according to Principle of Laboratory Animal Care (NIH Publication No. 85 -23, revised 1985) and the guidelines of the Peking University Animal Research Committee. Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation.", "Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation. Drugs were prepared in 2% DMSO in physiological saline and administered 4 h prior to virus exposure and continued daily for 5 days. All mice were observed daily for changes in weight and for any deaths.", "All mice were observed daily for changes in weight and for any deaths. Parameters for evaluation of antiviral activity included weight loss, reduction in mortality and/or increase in mean day to death (MDD) determined through 15 days. The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] .", "The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] . For convenience, the structure is named as NA hereafter. Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 .", "Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 . The docking and molecular dynamics (MD) simulations were performed by the general protocols in the Insight II 2005 software packages, consistent with the previous literatures [24, 26, 28, 35, [37] [38] [39] . During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K).", "During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K). The MD temperature was controlled by the velocity scaling thermostat [40] . Integrations of the classical equations of motion were achieved using the Verlet algorithm.", "Integrations of the classical equations of motion were achieved using the Verlet algorithm. The systems were solvated in a large sphere of TIP3P water molecules [40] with the radius of 35.0 Å, which is enough to hold the ensembles [40] . The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals.", "The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals. The interaction energies of patchouli alcohol with NA and the respective residues at the NA active site were calculated by the Docking module [35], over the 0.5~20.0 ns MD trajectories. All results are expressed as mean values ± standard deviations (SDs) (n = 3).", "All results are expressed as mean values ± standard deviations (SDs) (n = 3). The significance of difference was calculated by one-way analysis of variance, and values p < 0.001 were considered to be significant. In conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate.", "In conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate. Our results provide the promising information for the potential use of patchouli alcohol in the treatment of influenza A (H2N2) virus infectious disease. Further mechanistic studies on the anti-influenza A virus activity are needed to support this point of view." ]

[ "In the present study, the anti-influenza A (H2N2) virus activity of patchouli alcohol was studied in vitro, in vivo and in silico. The CC(50) of patchouli alcohol was above 20 µM. Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM.", "Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM. MTT assay showed that the inhibition by patchouli alcohol appears strongly after penetration of the virus into the cell. In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day.", "In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day. Flexible docking and molecular dynamic simulations indicated that patchouli alcohol was bound to the neuraminidase protein of influenza virus, with an interaction energy of –40.38 kcal mol(–1). The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process.", "The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process. Based on spatial and energetic criteria, patchouli alcohol interfered with the NA functions. Results presented here suggest that patchouli alcohol possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry.", "Results presented here suggest that patchouli alcohol possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry. Text: The influenza virus, which is one of the main causes of acute respiratory infections in humans, can lead to annual epidemics and infrequent pandemics. The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] .", "The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] . Today, their descendants continue to cause the majority of influenza infections in humans [3] . So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] .", "So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] . The release of new virions from the infected cell is a key step in the influenza life cycle and need neuraminidase (NA) to cleave the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue [6] . The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] .", "The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] . Consistent efforts have been devoted to the development of NA inhibitors, using the crystal structure of the N2 sub-type NA protein [8] [9] [10] [11] [12] [13] [14] [15] . Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] .", "Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] . However, with an increase in medical use, the oseltamivir-resistant strains have been found and probably lead to a large scale outbreak of novel pandemic flu [16, 17] . Patchouli alcohol ( Figure 1 ) has been well known for over a century.", "Patchouli alcohol ( Figure 1 ) has been well known for over a century. It is a major constituent of the pungent oil from the East Indian shrub Pogostemon cablin (Blanco) Benth, and widely used in fragrances. Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] .", "Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] . The essential oil is very appreciated for its characteristic pleasant and long lasting woody, earthy, and camphoraceous odor, as well as for its fixative properties, being suitable for use in soaps and cosmetic products [16, 17] . The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] .", "The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] . Moreover, the plant is widely used in Traditional Chinese Medicine as it presents various types of pharmacological activity according to the composition of the oil [16, 17] . Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] .", "Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] . To the best of our knowledge, the anti-influenza virus (H2N2) activities of patchouli alcohol have not been evaluated yet. Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model.", "Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model. On such basis, explicitly solvated docking and molecular dynamic (MD) methods were applied to investigative the binding mode involving patchouli alcohol with influenza virus NA protein. We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs.", "We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs. First the efficacy of patchouli alcohol on influenza A (H2N2) virus replication and cell viability were examined. CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK.", "CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK. The CC 50 of patchouli alcohol was above 20 mM, which indicated that patchouli alcohol did not affect the growth of MDCK (Table 1) . Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity.", "Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity. Moreover, patchouli alcohol was found to inhibit influenza A (H2N2) virus with an IC 50 of 4.03 ± 0.23 µM. Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96.", "Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96. It is reported that a SI of 4 or more is appropriate for an antiviral agent [18] , suggesting that patchouli alcohol can be judged to have anti-influenza A (H2N2) virus activity. Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM.", "Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM. Furthermore, it showed weak activity against B/Ibaraki/2/85 (IC 50 = 40.82 µM) [19] . With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol.", "With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol. Cells were pretreated with patchouli alcohol prior to virus infection (pretreatment cells), viruses were pretreated prior to infection (pretreatment virus), and patchouli alcohol was added during the adsorption period (adsorption) or after penetration of the viruses into cells (replication). Experiments were repeated independently three times and data presented are the average of three experiments.", "Experiments were repeated independently three times and data presented are the average of three experiments. The symbols * indicated very significant difference p < 0.01 with respect to other mode (pretreatment virus, adsorption and pretreatment cell). As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner.", "As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner. It showed best antiviral activity when added at a concentration of 8 µM during the replication period with inhibition of the viral replication of 97.68% ± 2.09% for influenza A (H2N2) at 72 h. However, no significant effect was detected when patchouli alcohol was used for pretreatment of cells or viruses or when patchouli alcohol was only added during the adsorption phase. These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell.", "These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell. Besides, biochemical studies have indicated that the bioactivity of NA protein is essential determinant after the replication of influenza A (H2N2) virus [20] [21] [22] . Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol.", "Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol. To evaluate the toxicity of patchouli alcohol, the mean value of body weight of mice in each group was statistically analyzed. The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05).", "The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05). Physiological status was observed in virus infection mice. Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc.", "Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc. In the mouse influenza model, viral infection leads to loss of body weight and high mortality. Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative to untreated infected (control) animals.", "Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative to untreated infected (control) animals. A comparison of efficacy of patchouli alcohol and oseltamivir in vivo mouse influenza model (oral treatment) showed that at a dose of 5 mg/kg/day, patchouli alcohol showed obvious protection against the influenza virus, as the mean day to death was detected as 11.8 ± 1.1 (Table 2) . When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8.", "When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8. Whereas oseltamivir at this dose level (1 mg/kg/day) showed 50% protection (measured by survivors/total) against the influenza virus. In the H2N2 infected control group, there were no survivors.", "In the H2N2 infected control group, there were no survivors. In view of both in vitro and in vivo data, we conclude that patchouli alcohol could be used in the treatment of human influenza virus infections. Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein.", "Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein. As the total energies and backbone root-mean-square-deviations (RMSD) in Figure 3 indicate, the energy-minimized patchouli alcohol-NA complex has been in equilibrium since about 0.5 ns, and then retains quite stable in the last 19.5 ns. It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] .", "It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] . Accordingly, the geometric and energetic analyses were made on the average structures of 0.5~20.0 ns MD trajectories, where the system has been already at equilibrium. The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ).", "The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ). As shown in Figure 4 , the patchouli alcohol was bound at the active site which also bound to oseltamivir and zanamivir [28] . As Figure 5 shows, the oxygen atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue.", "As Figure 5 shows, the oxygen atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue. The values of distances in Figure 6 further reveal that the docked complex remains rather stable throughout the simulation, with the average distances of Glu119:OE2patchouli alcohol:O and Tyr406:OH -patchouli alcohol:O less than 2.8 Å. The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) .", "The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) . Besides, patchouli alcohol was stabilized by residues Arg118, Asp151, Arg152, Trp178, Ala246, Glu276, Arg292, Asn294 and Gln347, especially residues Asp151, Arg152 and Glu276 ( Figure 5 and Table 3 ). As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] .", "As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] . The catalytic residues Asp151, Arg152 and Glu276 are crucial to the NA functions and the residues Glu119 and Tyr406 are important to stabilize the NA active sites [32, 33] . It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments.", "It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments. Patchouli alcohol matches with the NA active site and has an acceptable interaction energy. Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents.", "Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents. Patchouli alcohol and oseltamivir were obtained from Sigma Chemical Co. (St. Louis, MO, USA, purity > 99%) and was stored in glass vials with Teflon sealed caps at −20 ± 0.5 °C in the absence of light. MDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China).", "MDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China). The cells were grown in monolayer culture with Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin. The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent.", "The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent. Cells were plated out onto 96-well culture plates for cytotoxicity and anti-influenza assays, and propagated at 37 °C in an atmosphere of 5% CO 2 . The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China).", "The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China). Virus was routinely grown on MDCK cells. The stock cultures were prepared from supernatants of infected cells and stored at −80 °C. The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method.", "The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method. Briefly, cells were seeded on a microtiter plate in the absence or presence of various concentrations (20 µM -0.0098 µM) of patchouli alcohol (eight replicates) and incubated at 37 °C in a humidified atmosphere of 5% CO 2 for 72 h. The supernatants were discarded, washed with PBS twice and MTT reagent (5 mg/mL in PBS) was added to each well. After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min.", "After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min. After that the plates were read on an ELISA reader (Thermo Molecular Devices Co., Union City, USA) at 570/630 nm. The mean OD of the cell control wells was assigned a value of 100%.", "The mean OD of the cell control wells was assigned a value of 100%. The maximal non-toxic concentration (TD 0 ) and 50% cytotoxic concentration (CC 50 ) were calculated by linear regression analysis of the dose-response curves generated from the data. Inhibition of virus replication was measured by the MTT method.", "Inhibition of virus replication was measured by the MTT method. Serial dilution of the treated virus was adsorbed to the cells for 1 h at 37 °C. The residual inoculum was discared and infected cells were added with EMEM containing 2% FCS. Each assay was performed in eight replicates.", "Each assay was performed in eight replicates. After incubation for 72 h at 37 °C, the cultures were measured by MTT method as described above. The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves.", "The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves. Cells and viruses were incubated with patchouli alcohol at different stages during the viral infection cycle in order to determine the mode of antiviral action. Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells.", "Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells. Patchouli alcohol was always used at the nontoxic concentration. Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C.", "Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C. The compound was aspirated and cells were washed immediately before the influenza A (H2N2) inoculum was added. For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells.", "For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells. For analyzing the anti-influenza A (H2N2) inhibition during the adsorption period, the same amount of influenza A (H2N2) was mixed with the drug and added to the cells immediately. After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells.", "After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells. The effect of patchouli alcohol against influenza A (H2N2) was also tested during the replication period by adding it after adsorption, as typical performed in anti-influenza A (H2N2) susceptibility studies. Each assay was run in eight replicates.", "Each assay was run in eight replicates. Kunming mice, weighing 18-22 g (6 weeks of age) were purchased from Harbin Veterinary Research Institute Animal Co., Ltd. (Harbin, Heilongjiang, China) . First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline).", "First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline). The mice were orally administered with 10 mg/kg/dose patchouli alcohol, 2 mg/kg/dose patchouli alcohol or 2 mg/kg/dose oseltamivir (dissolved in 2% DMSO in physiological saline) one time daily for 7 days. The weight of mice was determined daily.", "The weight of mice was determined daily. We conducted procedures according to Principle of Laboratory Animal Care (NIH Publication No. 85 -23, revised 1985) and the guidelines of the Peking University Animal Research Committee. Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation.", "Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation. Drugs were prepared in 2% DMSO in physiological saline and administered 4 h prior to virus exposure and continued daily for 5 days. All mice were observed daily for changes in weight and for any deaths.", "All mice were observed daily for changes in weight and for any deaths. Parameters for evaluation of antiviral activity included weight loss, reduction in mortality and/or increase in mean day to death (MDD) determined through 15 days. The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] .", "The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] . For convenience, the structure is named as NA hereafter. Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 .", "Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 . The docking and molecular dynamics (MD) simulations were performed by the general protocols in the Insight II 2005 software packages, consistent with the previous literatures [24, 26, 28, 35, [37] [38] [39] . During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K).", "During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K). The MD temperature was controlled by the velocity scaling thermostat [40] . Integrations of the classical equations of motion were achieved using the Verlet algorithm.", "Integrations of the classical equations of motion were achieved using the Verlet algorithm. The systems were solvated in a large sphere of TIP3P water molecules [40] with the radius of 35.0 Å, which is enough to hold the ensembles [40] . The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals.", "The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals. The interaction energies of patchouli alcohol with NA and the respective residues at the NA active site were calculated by the Docking module [35], over the 0.5~20.0 ns MD trajectories. All results are expressed as mean values ± standard deviations (SDs) (n = 3).", "All results are expressed as mean values ± standard deviations (SDs) (n = 3). The significance of difference was calculated by one-way analysis of variance, and values p < 0.001 were considered to be significant. In conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate.", "In conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate. Our results provide the promising information for the potential use of patchouli alcohol in the treatment of influenza A (H2N2) virus infectious disease. Further mechanistic studies on the anti-influenza A virus activity are needed to support this point of view." ]

[ "In the present study, the anti-influenza A (H2N2) virus activity of patchouli alcohol was studied in vitro, in vivo and in silico. The CC(50) of patchouli alcohol was above 20 µM. Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM.", "Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM. MTT assay showed that the inhibition by patchouli alcohol appears strongly after penetration of the virus into the cell. In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day.", "In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day. Flexible docking and molecular dynamic simulations indicated that patchouli alcohol was bound to the neuraminidase protein of influenza virus, with an interaction energy of –40.38 kcal mol(–1). The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process.", "The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process. Based on spatial and energetic criteria, patchouli alcohol interfered with the NA functions. Results presented here suggest that patchouli alcohol possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry.", "Results presented here suggest that patchouli alcohol possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry. Text: The influenza virus, which is one of the main causes of acute respiratory infections in humans, can lead to annual epidemics and infrequent pandemics. The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] .", "The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] . Today, their descendants continue to cause the majority of influenza infections in humans [3] . So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] .", "So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] . The release of new virions from the infected cell is a key step in the influenza life cycle and need neuraminidase (NA) to cleave the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue [6] . The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] .", "The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] . Consistent efforts have been devoted to the development of NA inhibitors, using the crystal structure of the N2 sub-type NA protein [8] [9] [10] [11] [12] [13] [14] [15] . Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] .", "Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] . However, with an increase in medical use, the oseltamivir-resistant strains have been found and probably lead to a large scale outbreak of novel pandemic flu [16, 17] . Patchouli alcohol ( Figure 1 ) has been well known for over a century.", "Patchouli alcohol ( Figure 1 ) has been well known for over a century. It is a major constituent of the pungent oil from the East Indian shrub Pogostemon cablin (Blanco) Benth, and widely used in fragrances. Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] .", "Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] . The essential oil is very appreciated for its characteristic pleasant and long lasting woody, earthy, and camphoraceous odor, as well as for its fixative properties, being suitable for use in soaps and cosmetic products [16, 17] . The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] .", "The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] . Moreover, the plant is widely used in Traditional Chinese Medicine as it presents various types of pharmacological activity according to the composition of the oil [16, 17] . Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] .", "Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] . To the best of our knowledge, the anti-influenza virus (H2N2) activities of patchouli alcohol have not been evaluated yet. Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model.", "Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model. On such basis, explicitly solvated docking and molecular dynamic (MD) methods were applied to investigative the binding mode involving patchouli alcohol with influenza virus NA protein. We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs.", "We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs. First the efficacy of patchouli alcohol on influenza A (H2N2) virus replication and cell viability were examined. CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK.", "CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK. The CC 50 of patchouli alcohol was above 20 mM, which indicated that patchouli alcohol did not affect the growth of MDCK (Table 1) . Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity.", "Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity. Moreover, patchouli alcohol was found to inhibit influenza A (H2N2) virus with an IC 50 of 4.03 ± 0.23 µM. Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96.", "Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96. It is reported that a SI of 4 or more is appropriate for an antiviral agent [18] , suggesting that patchouli alcohol can be judged to have anti-influenza A (H2N2) virus activity. Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM.", "Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM. Furthermore, it showed weak activity against B/Ibaraki/2/85 (IC 50 = 40.82 µM) [19] . With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol.", "With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol. Cells were pretreated with patchouli alcohol prior to virus infection (pretreatment cells), viruses were pretreated prior to infection (pretreatment virus), and patchouli alcohol was added during the adsorption period (adsorption) or after penetration of the viruses into cells (replication). Experiments were repeated independently three times and data presented are the average of three experiments.", "Experiments were repeated independently three times and data presented are the average of three experiments. The symbols * indicated very significant difference p < 0.01 with respect to other mode (pretreatment virus, adsorption and pretreatment cell). As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner.", "As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner. It showed best antiviral activity when added at a concentration of 8 µM during the replication period with inhibition of the viral replication of 97.68% ± 2.09% for influenza A (H2N2) at 72 h. However, no significant effect was detected when patchouli alcohol was used for pretreatment of cells or viruses or when patchouli alcohol was only added during the adsorption phase. These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell.", "These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell. Besides, biochemical studies have indicated that the bioactivity of NA protein is essential determinant after the replication of influenza A (H2N2) virus [20] [21] [22] . Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol.", "Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol. To evaluate the toxicity of patchouli alcohol, the mean value of body weight of mice in each group was statistically analyzed. The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05).", "The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05). Physiological status was observed in virus infection mice. Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc.", "Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc. In the mouse influenza model, viral infection leads to loss of body weight and high mortality. Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative to untreated infected (control) animals.", "Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative to untreated infected (control) animals. A comparison of efficacy of patchouli alcohol and oseltamivir in vivo mouse influenza model (oral treatment) showed that at a dose of 5 mg/kg/day, patchouli alcohol showed obvious protection against the influenza virus, as the mean day to death was detected as 11.8 ± 1.1 (Table 2) . When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8.", "When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8. Whereas oseltamivir at this dose level (1 mg/kg/day) showed 50% protection (measured by survivors/total) against the influenza virus. In the H2N2 infected control group, there were no survivors.", "In the H2N2 infected control group, there were no survivors. In view of both in vitro and in vivo data, we conclude that patchouli alcohol could be used in the treatment of human influenza virus infections. Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein.", "Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein. As the total energies and backbone root-mean-square-deviations (RMSD) in Figure 3 indicate, the energy-minimized patchouli alcohol-NA complex has been in equilibrium since about 0.5 ns, and then retains quite stable in the last 19.5 ns. It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] .", "It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] . Accordingly, the geometric and energetic analyses were made on the average structures of 0.5~20.0 ns MD trajectories, where the system has been already at equilibrium. The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ).", "The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ). As shown in Figure 4 , the patchouli alcohol was bound at the active site which also bound to oseltamivir and zanamivir [28] . As Figure 5 shows, the oxygen atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue.", "As Figure 5 shows, the oxygen atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue. The values of distances in Figure 6 further reveal that the docked complex remains rather stable throughout the simulation, with the average distances of Glu119:OE2patchouli alcohol:O and Tyr406:OH -patchouli alcohol:O less than 2.8 Å. The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) .", "The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) . Besides, patchouli alcohol was stabilized by residues Arg118, Asp151, Arg152, Trp178, Ala246, Glu276, Arg292, Asn294 and Gln347, especially residues Asp151, Arg152 and Glu276 ( Figure 5 and Table 3 ). As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] .", "As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] . The catalytic residues Asp151, Arg152 and Glu276 are crucial to the NA functions and the residues Glu119 and Tyr406 are important to stabilize the NA active sites [32, 33] . It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments.", "It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments. Patchouli alcohol matches with the NA active site and has an acceptable interaction energy. Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents.", "Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents. Patchouli alcohol and oseltamivir were obtained from Sigma Chemical Co. (St. Louis, MO, USA, purity > 99%) and was stored in glass vials with Teflon sealed caps at −20 ± 0.5 °C in the absence of light. MDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China).", "MDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China). The cells were grown in monolayer culture with Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin. The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent.", "The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent. Cells were plated out onto 96-well culture plates for cytotoxicity and anti-influenza assays, and propagated at 37 °C in an atmosphere of 5% CO 2 . The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China).", "The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China). Virus was routinely grown on MDCK cells. The stock cultures were prepared from supernatants of infected cells and stored at −80 °C. The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method.", "The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method. Briefly, cells were seeded on a microtiter plate in the absence or presence of various concentrations (20 µM -0.0098 µM) of patchouli alcohol (eight replicates) and incubated at 37 °C in a humidified atmosphere of 5% CO 2 for 72 h. The supernatants were discarded, washed with PBS twice and MTT reagent (5 mg/mL in PBS) was added to each well. After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min.", "After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min. After that the plates were read on an ELISA reader (Thermo Molecular Devices Co., Union City, USA) at 570/630 nm. The mean OD of the cell control wells was assigned a value of 100%.", "The mean OD of the cell control wells was assigned a value of 100%. The maximal non-toxic concentration (TD 0 ) and 50% cytotoxic concentration (CC 50 ) were calculated by linear regression analysis of the dose-response curves generated from the data. Inhibition of virus replication was measured by the MTT method.", "Inhibition of virus replication was measured by the MTT method. Serial dilution of the treated virus was adsorbed to the cells for 1 h at 37 °C. The residual inoculum was discared and infected cells were added with EMEM containing 2% FCS. Each assay was performed in eight replicates.", "Each assay was performed in eight replicates. After incubation for 72 h at 37 °C, the cultures were measured by MTT method as described above. The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves.", "The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves. Cells and viruses were incubated with patchouli alcohol at different stages during the viral infection cycle in order to determine the mode of antiviral action. Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells.", "Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells. Patchouli alcohol was always used at the nontoxic concentration. Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C.", "Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C. The compound was aspirated and cells were washed immediately before the influenza A (H2N2) inoculum was added. For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells.", "For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells. For analyzing the anti-influenza A (H2N2) inhibition during the adsorption period, the same amount of influenza A (H2N2) was mixed with the drug and added to the cells immediately. After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells.", "After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells. The effect of patchouli alcohol against influenza A (H2N2) was also tested during the replication period by adding it after adsorption, as typical performed in anti-influenza A (H2N2) susceptibility studies. Each assay was run in eight replicates.", "Each assay was run in eight replicates. Kunming mice, weighing 18-22 g (6 weeks of age) were purchased from Harbin Veterinary Research Institute Animal Co., Ltd. (Harbin, Heilongjiang, China) . First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline).", "First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline). The mice were orally administered with 10 mg/kg/dose patchouli alcohol, 2 mg/kg/dose patchouli alcohol or 2 mg/kg/dose oseltamivir (dissolved in 2% DMSO in physiological saline) one time daily for 7 days. The weight of mice was determined daily.", "The weight of mice was determined daily. We conducted procedures according to Principle of Laboratory Animal Care (NIH Publication No. 85 -23, revised 1985) and the guidelines of the Peking University Animal Research Committee. Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation.", "Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation. Drugs were prepared in 2% DMSO in physiological saline and administered 4 h prior to virus exposure and continued daily for 5 days. All mice were observed daily for changes in weight and for any deaths.", "All mice were observed daily for changes in weight and for any deaths. Parameters for evaluation of antiviral activity included weight loss, reduction in mortality and/or increase in mean day to death (MDD) determined through 15 days. The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] .", "The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] . For convenience, the structure is named as NA hereafter. Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 .", "Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 . The docking and molecular dynamics (MD) simulations were performed by the general protocols in the Insight II 2005 software packages, consistent with the previous literatures [24, 26, 28, 35, [37] [38] [39] . During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K).", "During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K). The MD temperature was controlled by the velocity scaling thermostat [40] . Integrations of the classical equations of motion were achieved using the Verlet algorithm.", "Integrations of the classical equations of motion were achieved using the Verlet algorithm. The systems were solvated in a large sphere of TIP3P water molecules [40] with the radius of 35.0 Å, which is enough to hold the ensembles [40] . The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals.", "The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals. The interaction energies of patchouli alcohol with NA and the respective residues at the NA active site were calculated by the Docking module [35], over the 0.5~20.0 ns MD trajectories. All results are expressed as mean values ± standard deviations (SDs) (n = 3).", "All results are expressed as mean values ± standard deviations (SDs) (n = 3). The significance of difference was calculated by one-way analysis of variance, and values p < 0.001 were considered to be significant. In conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate.", "In conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate. Our results provide the promising information for the potential use of patchouli alcohol in the treatment of influenza A (H2N2) virus infectious disease. Further mechanistic studies on the anti-influenza A virus activity are needed to support this point of view." ]

[ "In the present study, the anti-influenza A (H2N2) virus activity of patchouli alcohol was studied in vitro, in vivo and in silico. The CC(50) of patchouli alcohol was above 20 µM. Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM.", "Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM. MTT assay showed that the inhibition by patchouli alcohol appears strongly after penetration of the virus into the cell. In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day.", "In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day. Flexible docking and molecular dynamic simulations indicated that patchouli alcohol was bound to the neuraminidase protein of influenza virus, with an interaction energy of –40.38 kcal mol(–1). The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process.", "The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process. Based on spatial and energetic criteria, patchouli alcohol interfered with the NA functions. Results presented here suggest that patchouli alcohol possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry.", "Results presented here suggest that patchouli alcohol possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry. Text: The influenza virus, which is one of the main causes of acute respiratory infections in humans, can lead to annual epidemics and infrequent pandemics. The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] .", "The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] . Today, their descendants continue to cause the majority of influenza infections in humans [3] . So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] .", "So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] . The release of new virions from the infected cell is a key step in the influenza life cycle and need neuraminidase (NA) to cleave the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue [6] . The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] .", "The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] . Consistent efforts have been devoted to the development of NA inhibitors, using the crystal structure of the N2 sub-type NA protein [8] [9] [10] [11] [12] [13] [14] [15] . Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] .", "Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] . However, with an increase in medical use, the oseltamivir-resistant strains have been found and probably lead to a large scale outbreak of novel pandemic flu [16, 17] . Patchouli alcohol ( Figure 1 ) has been well known for over a century.", "Patchouli alcohol ( Figure 1 ) has been well known for over a century. It is a major constituent of the pungent oil from the East Indian shrub Pogostemon cablin (Blanco) Benth, and widely used in fragrances. Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] .", "Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] . The essential oil is very appreciated for its characteristic pleasant and long lasting woody, earthy, and camphoraceous odor, as well as for its fixative properties, being suitable for use in soaps and cosmetic products [16, 17] . The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] .", "The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] . Moreover, the plant is widely used in Traditional Chinese Medicine as it presents various types of pharmacological activity according to the composition of the oil [16, 17] . Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] .", "Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] . To the best of our knowledge, the anti-influenza virus (H2N2) activities of patchouli alcohol have not been evaluated yet. Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model.", "Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model. On such basis, explicitly solvated docking and molecular dynamic (MD) methods were applied to investigative the binding mode involving patchouli alcohol with influenza virus NA protein. We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs.", "We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs. First the efficacy of patchouli alcohol on influenza A (H2N2) virus replication and cell viability were examined. CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK.", "CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK. The CC 50 of patchouli alcohol was above 20 mM, which indicated that patchouli alcohol did not affect the growth of MDCK (Table 1) . Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity.", "Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity. Moreover, patchouli alcohol was found to inhibit influenza A (H2N2) virus with an IC 50 of 4.03 ± 0.23 µM. Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96.", "Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96. It is reported that a SI of 4 or more is appropriate for an antiviral agent [18] , suggesting that patchouli alcohol can be judged to have anti-influenza A (H2N2) virus activity. Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM.", "Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM. Furthermore, it showed weak activity against B/Ibaraki/2/85 (IC 50 = 40.82 µM) [19] . With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol.", "With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol. Cells were pretreated with patchouli alcohol prior to virus infection (pretreatment cells), viruses were pretreated prior to infection (pretreatment virus), and patchouli alcohol was added during the adsorption period (adsorption) or after penetration of the viruses into cells (replication). Experiments were repeated independently three times and data presented are the average of three experiments.", "Experiments were repeated independently three times and data presented are the average of three experiments. The symbols * indicated very significant difference p < 0.01 with respect to other mode (pretreatment virus, adsorption and pretreatment cell). As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner.", "As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner. It showed best antiviral activity when added at a concentration of 8 µM during the replication period with inhibition of the viral replication of 97.68% ± 2.09% for influenza A (H2N2) at 72 h. However, no significant effect was detected when patchouli alcohol was used for pretreatment of cells or viruses or when patchouli alcohol was only added during the adsorption phase. These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell.", "These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell. Besides, biochemical studies have indicated that the bioactivity of NA protein is essential determinant after the replication of influenza A (H2N2) virus [20] [21] [22] . Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol.", "Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol. To evaluate the toxicity of patchouli alcohol, the mean value of body weight of mice in each group was statistically analyzed. The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05).", "The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05). Physiological status was observed in virus infection mice. Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc.", "Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc. In the mouse influenza model, viral infection leads to loss of body weight and high mortality. Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative to untreated infected (control) animals.", "Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative to untreated infected (control) animals. A comparison of efficacy of patchouli alcohol and oseltamivir in vivo mouse influenza model (oral treatment) showed that at a dose of 5 mg/kg/day, patchouli alcohol showed obvious protection against the influenza virus, as the mean day to death was detected as 11.8 ± 1.1 (Table 2) . When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8.", "When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8. Whereas oseltamivir at this dose level (1 mg/kg/day) showed 50% protection (measured by survivors/total) against the influenza virus. In the H2N2 infected control group, there were no survivors.", "In the H2N2 infected control group, there were no survivors. In view of both in vitro and in vivo data, we conclude that patchouli alcohol could be used in the treatment of human influenza virus infections. Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein.", "Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein. As the total energies and backbone root-mean-square-deviations (RMSD) in Figure 3 indicate, the energy-minimized patchouli alcohol-NA complex has been in equilibrium since about 0.5 ns, and then retains quite stable in the last 19.5 ns. It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] .", "It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] . Accordingly, the geometric and energetic analyses were made on the average structures of 0.5~20.0 ns MD trajectories, where the system has been already at equilibrium. The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ).", "The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ). As shown in Figure 4 , the patchouli alcohol was bound at the active site which also bound to oseltamivir and zanamivir [28] . As Figure 5 shows, the oxygen atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue.", "As Figure 5 shows, the oxygen atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue. The values of distances in Figure 6 further reveal that the docked complex remains rather stable throughout the simulation, with the average distances of Glu119:OE2patchouli alcohol:O and Tyr406:OH -patchouli alcohol:O less than 2.8 Å. The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) .", "The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) . Besides, patchouli alcohol was stabilized by residues Arg118, Asp151, Arg152, Trp178, Ala246, Glu276, Arg292, Asn294 and Gln347, especially residues Asp151, Arg152 and Glu276 ( Figure 5 and Table 3 ). As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] .", "As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] . The catalytic residues Asp151, Arg152 and Glu276 are crucial to the NA functions and the residues Glu119 and Tyr406 are important to stabilize the NA active sites [32, 33] . It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments.", "It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments. Patchouli alcohol matches with the NA active site and has an acceptable interaction energy. Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents.", "Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents. Patchouli alcohol and oseltamivir were obtained from Sigma Chemical Co. (St. Louis, MO, USA, purity > 99%) and was stored in glass vials with Teflon sealed caps at −20 ± 0.5 °C in the absence of light. MDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China).", "MDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China). The cells were grown in monolayer culture with Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin. The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent.", "The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent. Cells were plated out onto 96-well culture plates for cytotoxicity and anti-influenza assays, and propagated at 37 °C in an atmosphere of 5% CO 2 . The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China).", "The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China). Virus was routinely grown on MDCK cells. The stock cultures were prepared from supernatants of infected cells and stored at −80 °C. The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method.", "The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method. Briefly, cells were seeded on a microtiter plate in the absence or presence of various concentrations (20 µM -0.0098 µM) of patchouli alcohol (eight replicates) and incubated at 37 °C in a humidified atmosphere of 5% CO 2 for 72 h. The supernatants were discarded, washed with PBS twice and MTT reagent (5 mg/mL in PBS) was added to each well. After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min.", "After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min. After that the plates were read on an ELISA reader (Thermo Molecular Devices Co., Union City, USA) at 570/630 nm. The mean OD of the cell control wells was assigned a value of 100%.", "The mean OD of the cell control wells was assigned a value of 100%. The maximal non-toxic concentration (TD 0 ) and 50% cytotoxic concentration (CC 50 ) were calculated by linear regression analysis of the dose-response curves generated from the data. Inhibition of virus replication was measured by the MTT method.", "Inhibition of virus replication was measured by the MTT method. Serial dilution of the treated virus was adsorbed to the cells for 1 h at 37 °C. The residual inoculum was discared and infected cells were added with EMEM containing 2% FCS. Each assay was performed in eight replicates.", "Each assay was performed in eight replicates. After incubation for 72 h at 37 °C, the cultures were measured by MTT method as described above. The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves.", "The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves. Cells and viruses were incubated with patchouli alcohol at different stages during the viral infection cycle in order to determine the mode of antiviral action. Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells.", "Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells. Patchouli alcohol was always used at the nontoxic concentration. Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C.", "Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C. The compound was aspirated and cells were washed immediately before the influenza A (H2N2) inoculum was added. For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells.", "For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells. For analyzing the anti-influenza A (H2N2) inhibition during the adsorption period, the same amount of influenza A (H2N2) was mixed with the drug and added to the cells immediately. After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells.", "After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells. The effect of patchouli alcohol against influenza A (H2N2) was also tested during the replication period by adding it after adsorption, as typical performed in anti-influenza A (H2N2) susceptibility studies. Each assay was run in eight replicates.", "Each assay was run in eight replicates. Kunming mice, weighing 18-22 g (6 weeks of age) were purchased from Harbin Veterinary Research Institute Animal Co., Ltd. (Harbin, Heilongjiang, China) . First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline).", "First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline). The mice were orally administered with 10 mg/kg/dose patchouli alcohol, 2 mg/kg/dose patchouli alcohol or 2 mg/kg/dose oseltamivir (dissolved in 2% DMSO in physiological saline) one time daily for 7 days. The weight of mice was determined daily.", "The weight of mice was determined daily. We conducted procedures according to Principle of Laboratory Animal Care (NIH Publication No. 85 -23, revised 1985) and the guidelines of the Peking University Animal Research Committee. Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation.", "Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation. Drugs were prepared in 2% DMSO in physiological saline and administered 4 h prior to virus exposure and continued daily for 5 days. All mice were observed daily for changes in weight and for any deaths.", "All mice were observed daily for changes in weight and for any deaths. Parameters for evaluation of antiviral activity included weight loss, reduction in mortality and/or increase in mean day to death (MDD) determined through 15 days. The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] .", "The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] . For convenience, the structure is named as NA hereafter. Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 .", "Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 . The docking and molecular dynamics (MD) simulations were performed by the general protocols in the Insight II 2005 software packages, consistent with the previous literatures [24, 26, 28, 35, [37] [38] [39] . During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K).", "During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K). The MD temperature was controlled by the velocity scaling thermostat [40] . Integrations of the classical equations of motion were achieved using the Verlet algorithm.", "Integrations of the classical equations of motion were achieved using the Verlet algorithm. The systems were solvated in a large sphere of TIP3P water molecules [40] with the radius of 35.0 Å, which is enough to hold the ensembles [40] . The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals.", "The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals. The interaction energies of patchouli alcohol with NA and the respective residues at the NA active site were calculated by the Docking module [35], over the 0.5~20.0 ns MD trajectories. All results are expressed as mean values ± standard deviations (SDs) (n = 3).", "All results are expressed as mean values ± standard deviations (SDs) (n = 3). The significance of difference was calculated by one-way analysis of variance, and values p < 0.001 were considered to be significant. In conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate.", "In conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate. Our results provide the promising information for the potential use of patchouli alcohol in the treatment of influenza A (H2N2) virus infectious disease. Further mechanistic studies on the anti-influenza A virus activity are needed to support this point of view." ]

[ "In the present study, the anti-influenza A (H2N2) virus activity of patchouli alcohol was studied in vitro, in vivo and in silico. The CC(50) of patchouli alcohol was above 20 µM. Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM.", "Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM. MTT assay showed that the inhibition by patchouli alcohol appears strongly after penetration of the virus into the cell. In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day.", "In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day. Flexible docking and molecular dynamic simulations indicated that patchouli alcohol was bound to the neuraminidase protein of influenza virus, with an interaction energy of –40.38 kcal mol(–1). The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process.", "The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process. Based on spatial and energetic criteria, patchouli alcohol interfered with the NA functions. Results presented here suggest that patchouli alcohol possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry.", "Results presented here suggest that patchouli alcohol possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry. Text: The influenza virus, which is one of the main causes of acute respiratory infections in humans, can lead to annual epidemics and infrequent pandemics. The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] .", "The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] . Today, their descendants continue to cause the majority of influenza infections in humans [3] . So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] .", "So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] . The release of new virions from the infected cell is a key step in the influenza life cycle and need neuraminidase (NA) to cleave the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue [6] . The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] .", "The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] . Consistent efforts have been devoted to the development of NA inhibitors, using the crystal structure of the N2 sub-type NA protein [8] [9] [10] [11] [12] [13] [14] [15] . Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] .", "Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] . However, with an increase in medical use, the oseltamivir-resistant strains have been found and probably lead to a large scale outbreak of novel pandemic flu [16, 17] . Patchouli alcohol ( Figure 1 ) has been well known for over a century.", "Patchouli alcohol ( Figure 1 ) has been well known for over a century. It is a major constituent of the pungent oil from the East Indian shrub Pogostemon cablin (Blanco) Benth, and widely used in fragrances. Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] .", "Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] . The essential oil is very appreciated for its characteristic pleasant and long lasting woody, earthy, and camphoraceous odor, as well as for its fixative properties, being suitable for use in soaps and cosmetic products [16, 17] . The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] .", "The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] . Moreover, the plant is widely used in Traditional Chinese Medicine as it presents various types of pharmacological activity according to the composition of the oil [16, 17] . Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] .", "Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] . To the best of our knowledge, the anti-influenza virus (H2N2) activities of patchouli alcohol have not been evaluated yet. Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model.", "Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model. On such basis, explicitly solvated docking and molecular dynamic (MD) methods were applied to investigative the binding mode involving patchouli alcohol with influenza virus NA protein. We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs.", "We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs. First the efficacy of patchouli alcohol on influenza A (H2N2) virus replication and cell viability were examined. CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK.", "CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK. The CC 50 of patchouli alcohol was above 20 mM, which indicated that patchouli alcohol did not affect the growth of MDCK (Table 1) . Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity.", "Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity. Moreover, patchouli alcohol was found to inhibit influenza A (H2N2) virus with an IC 50 of 4.03 ± 0.23 µM. Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96.", "Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96. It is reported that a SI of 4 or more is appropriate for an antiviral agent [18] , suggesting that patchouli alcohol can be judged to have anti-influenza A (H2N2) virus activity. Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM.", "Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM. Furthermore, it showed weak activity against B/Ibaraki/2/85 (IC 50 = 40.82 µM) [19] . With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol.", "With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol. Cells were pretreated with patchouli alcohol prior to virus infection (pretreatment cells), viruses were pretreated prior to infection (pretreatment virus), and patchouli alcohol was added during the adsorption period (adsorption) or after penetration of the viruses into cells (replication). Experiments were repeated independently three times and data presented are the average of three experiments.", "Experiments were repeated independently three times and data presented are the average of three experiments. The symbols * indicated very significant difference p < 0.01 with respect to other mode (pretreatment virus, adsorption and pretreatment cell). As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner.", "As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner. It showed best antiviral activity when added at a concentration of 8 µM during the replication period with inhibition of the viral replication of 97.68% ± 2.09% for influenza A (H2N2) at 72 h. However, no significant effect was detected when patchouli alcohol was used for pretreatment of cells or viruses or when patchouli alcohol was only added during the adsorption phase. These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell.", "These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell. Besides, biochemical studies have indicated that the bioactivity of NA protein is essential determinant after the replication of influenza A (H2N2) virus [20] [21] [22] . Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol.", "Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol. To evaluate the toxicity of patchouli alcohol, the mean value of body weight of mice in each group was statistically analyzed. The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05).", "The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05). Physiological status was observed in virus infection mice. Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc.", "Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc. In the mouse influenza model, viral infection leads to loss of body weight and high mortality. Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative to untreated infected (control) animals.", "Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative to untreated infected (control) animals. A comparison of efficacy of patchouli alcohol and oseltamivir in vivo mouse influenza model (oral treatment) showed that at a dose of 5 mg/kg/day, patchouli alcohol showed obvious protection against the influenza virus, as the mean day to death was detected as 11.8 ± 1.1 (Table 2) . When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8.", "When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8. Whereas oseltamivir at this dose level (1 mg/kg/day) showed 50% protection (measured by survivors/total) against the influenza virus. In the H2N2 infected control group, there were no survivors.", "In the H2N2 infected control group, there were no survivors. In view of both in vitro and in vivo data, we conclude that patchouli alcohol could be used in the treatment of human influenza virus infections. Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein.", "Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein. As the total energies and backbone root-mean-square-deviations (RMSD) in Figure 3 indicate, the energy-minimized patchouli alcohol-NA complex has been in equilibrium since about 0.5 ns, and then retains quite stable in the last 19.5 ns. It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] .", "It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] . Accordingly, the geometric and energetic analyses were made on the average structures of 0.5~20.0 ns MD trajectories, where the system has been already at equilibrium. The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ).", "The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ). As shown in Figure 4 , the patchouli alcohol was bound at the active site which also bound to oseltamivir and zanamivir [28] . As Figure 5 shows, the oxygen atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue.", "As Figure 5 shows, the oxygen atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue. The values of distances in Figure 6 further reveal that the docked complex remains rather stable throughout the simulation, with the average distances of Glu119:OE2patchouli alcohol:O and Tyr406:OH -patchouli alcohol:O less than 2.8 Å. The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) .", "The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) . Besides, patchouli alcohol was stabilized by residues Arg118, Asp151, Arg152, Trp178, Ala246, Glu276, Arg292, Asn294 and Gln347, especially residues Asp151, Arg152 and Glu276 ( Figure 5 and Table 3 ). As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] .", "As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] . The catalytic residues Asp151, Arg152 and Glu276 are crucial to the NA functions and the residues Glu119 and Tyr406 are important to stabilize the NA active sites [32, 33] . It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments.", "It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments. Patchouli alcohol matches with the NA active site and has an acceptable interaction energy. Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents.", "Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents. Patchouli alcohol and oseltamivir were obtained from Sigma Chemical Co. (St. Louis, MO, USA, purity > 99%) and was stored in glass vials with Teflon sealed caps at −20 ± 0.5 °C in the absence of light. MDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China).", "MDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China). The cells were grown in monolayer culture with Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin. The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent.", "The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent. Cells were plated out onto 96-well culture plates for cytotoxicity and anti-influenza assays, and propagated at 37 °C in an atmosphere of 5% CO 2 . The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China).", "The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China). Virus was routinely grown on MDCK cells. The stock cultures were prepared from supernatants of infected cells and stored at −80 °C. The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method.", "The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method. Briefly, cells were seeded on a microtiter plate in the absence or presence of various concentrations (20 µM -0.0098 µM) of patchouli alcohol (eight replicates) and incubated at 37 °C in a humidified atmosphere of 5% CO 2 for 72 h. The supernatants were discarded, washed with PBS twice and MTT reagent (5 mg/mL in PBS) was added to each well. After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min.", "After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min. After that the plates were read on an ELISA reader (Thermo Molecular Devices Co., Union City, USA) at 570/630 nm. The mean OD of the cell control wells was assigned a value of 100%.", "The mean OD of the cell control wells was assigned a value of 100%. The maximal non-toxic concentration (TD 0 ) and 50% cytotoxic concentration (CC 50 ) were calculated by linear regression analysis of the dose-response curves generated from the data. Inhibition of virus replication was measured by the MTT method.", "Inhibition of virus replication was measured by the MTT method. Serial dilution of the treated virus was adsorbed to the cells for 1 h at 37 °C. The residual inoculum was discared and infected cells were added with EMEM containing 2% FCS. Each assay was performed in eight replicates.", "Each assay was performed in eight replicates. After incubation for 72 h at 37 °C, the cultures were measured by MTT method as described above. The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves.", "The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves. Cells and viruses were incubated with patchouli alcohol at different stages during the viral infection cycle in order to determine the mode of antiviral action. Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells.", "Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells. Patchouli alcohol was always used at the nontoxic concentration. Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C.", "Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C. The compound was aspirated and cells were washed immediately before the influenza A (H2N2) inoculum was added. For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells.", "For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells. For analyzing the anti-influenza A (H2N2) inhibition during the adsorption period, the same amount of influenza A (H2N2) was mixed with the drug and added to the cells immediately. After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells.", "After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells. The effect of patchouli alcohol against influenza A (H2N2) was also tested during the replication period by adding it after adsorption, as typical performed in anti-influenza A (H2N2) susceptibility studies. Each assay was run in eight replicates.", "Each assay was run in eight replicates. Kunming mice, weighing 18-22 g (6 weeks of age) were purchased from Harbin Veterinary Research Institute Animal Co., Ltd. (Harbin, Heilongjiang, China) . First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline).", "First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline). The mice were orally administered with 10 mg/kg/dose patchouli alcohol, 2 mg/kg/dose patchouli alcohol or 2 mg/kg/dose oseltamivir (dissolved in 2% DMSO in physiological saline) one time daily for 7 days. The weight of mice was determined daily.", "The weight of mice was determined daily. We conducted procedures according to Principle of Laboratory Animal Care (NIH Publication No. 85 -23, revised 1985) and the guidelines of the Peking University Animal Research Committee. Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation.", "Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation. Drugs were prepared in 2% DMSO in physiological saline and administered 4 h prior to virus exposure and continued daily for 5 days. All mice were observed daily for changes in weight and for any deaths.", "All mice were observed daily for changes in weight and for any deaths. Parameters for evaluation of antiviral activity included weight loss, reduction in mortality and/or increase in mean day to death (MDD) determined through 15 days. The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] .", "The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] . For convenience, the structure is named as NA hereafter. Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 .", "Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 . The docking and molecular dynamics (MD) simulations were performed by the general protocols in the Insight II 2005 software packages, consistent with the previous literatures [24, 26, 28, 35, [37] [38] [39] . During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K).", "During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K). The MD temperature was controlled by the velocity scaling thermostat [40] . Integrations of the classical equations of motion were achieved using the Verlet algorithm.", "Integrations of the classical equations of motion were achieved using the Verlet algorithm. The systems were solvated in a large sphere of TIP3P water molecules [40] with the radius of 35.0 Å, which is enough to hold the ensembles [40] . The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals.", "The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals. The interaction energies of patchouli alcohol with NA and the respective residues at the NA active site were calculated by the Docking module [35], over the 0.5~20.0 ns MD trajectories. All results are expressed as mean values ± standard deviations (SDs) (n = 3).", "All results are expressed as mean values ± standard deviations (SDs) (n = 3). The significance of difference was calculated by one-way analysis of variance, and values p < 0.001 were considered to be significant. In conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate.", "In conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate. Our results provide the promising information for the potential use of patchouli alcohol in the treatment of influenza A (H2N2) virus infectious disease. Further mechanistic studies on the anti-influenza A virus activity are needed to support this point of view." ]

[ "In the present study, the anti-influenza A (H2N2) virus activity of patchouli alcohol was studied in vitro, in vivo and in silico. The CC(50) of patchouli alcohol was above 20 µM. Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM.", "Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM. MTT assay showed that the inhibition by patchouli alcohol appears strongly after penetration of the virus into the cell. In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day.", "In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day. Flexible docking and molecular dynamic simulations indicated that patchouli alcohol was bound to the neuraminidase protein of influenza virus, with an interaction energy of –40.38 kcal mol(–1). The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process.", "The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process. Based on spatial and energetic criteria, patchouli alcohol interfered with the NA functions. Results presented here suggest that patchouli alcohol possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry.", "Results presented here suggest that patchouli alcohol possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry. Text: The influenza virus, which is one of the main causes of acute respiratory infections in humans, can lead to annual epidemics and infrequent pandemics. The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] .", "The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] . Today, their descendants continue to cause the majority of influenza infections in humans [3] . So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] .", "So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] . The release of new virions from the infected cell is a key step in the influenza life cycle and need neuraminidase (NA) to cleave the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue [6] . The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] .", "The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] . Consistent efforts have been devoted to the development of NA inhibitors, using the crystal structure of the N2 sub-type NA protein [8] [9] [10] [11] [12] [13] [14] [15] . Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] .", "Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] . However, with an increase in medical use, the oseltamivir-resistant strains have been found and probably lead to a large scale outbreak of novel pandemic flu [16, 17] . Patchouli alcohol ( Figure 1 ) has been well known for over a century.", "Patchouli alcohol ( Figure 1 ) has been well known for over a century. It is a major constituent of the pungent oil from the East Indian shrub Pogostemon cablin (Blanco) Benth, and widely used in fragrances. Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] .", "Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] . The essential oil is very appreciated for its characteristic pleasant and long lasting woody, earthy, and camphoraceous odor, as well as for its fixative properties, being suitable for use in soaps and cosmetic products [16, 17] . The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] .", "The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] . Moreover, the plant is widely used in Traditional Chinese Medicine as it presents various types of pharmacological activity according to the composition of the oil [16, 17] . Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] .", "Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] . To the best of our knowledge, the anti-influenza virus (H2N2) activities of patchouli alcohol have not been evaluated yet. Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model.", "Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model. On such basis, explicitly solvated docking and molecular dynamic (MD) methods were applied to investigative the binding mode involving patchouli alcohol with influenza virus NA protein. We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs.", "We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs. First the efficacy of patchouli alcohol on influenza A (H2N2) virus replication and cell viability were examined. CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK.", "CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK. The CC 50 of patchouli alcohol was above 20 mM, which indicated that patchouli alcohol did not affect the growth of MDCK (Table 1) . Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity.", "Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity. Moreover, patchouli alcohol was found to inhibit influenza A (H2N2) virus with an IC 50 of 4.03 ± 0.23 µM. Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96.", "Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96. It is reported that a SI of 4 or more is appropriate for an antiviral agent [18] , suggesting that patchouli alcohol can be judged to have anti-influenza A (H2N2) virus activity. Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM.", "Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM. Furthermore, it showed weak activity against B/Ibaraki/2/85 (IC 50 = 40.82 µM) [19] . With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol.", "With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol. Cells were pretreated with patchouli alcohol prior to virus infection (pretreatment cells), viruses were pretreated prior to infection (pretreatment virus), and patchouli alcohol was added during the adsorption period (adsorption) or after penetration of the viruses into cells (replication). Experiments were repeated independently three times and data presented are the average of three experiments.", "Experiments were repeated independently three times and data presented are the average of three experiments. The symbols * indicated very significant difference p < 0.01 with respect to other mode (pretreatment virus, adsorption and pretreatment cell). As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner.", "As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner. It showed best antiviral activity when added at a concentration of 8 µM during the replication period with inhibition of the viral replication of 97.68% ± 2.09% for influenza A (H2N2) at 72 h. However, no significant effect was detected when patchouli alcohol was used for pretreatment of cells or viruses or when patchouli alcohol was only added during the adsorption phase. These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell.", "These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell. Besides, biochemical studies have indicated that the bioactivity of NA protein is essential determinant after the replication of influenza A (H2N2) virus [20] [21] [22] . Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol.", "Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol. To evaluate the toxicity of patchouli alcohol, the mean value of body weight of mice in each group was statistically analyzed. The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05).", "The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05). Physiological status was observed in virus infection mice. Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc.", "Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc. In the mouse influenza model, viral infection leads to loss of body weight and high mortality. Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative to untreated infected (control) animals.", "Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative to untreated infected (control) animals. A comparison of efficacy of patchouli alcohol and oseltamivir in vivo mouse influenza model (oral treatment) showed that at a dose of 5 mg/kg/day, patchouli alcohol showed obvious protection against the influenza virus, as the mean day to death was detected as 11.8 ± 1.1 (Table 2) . When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8.", "When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8. Whereas oseltamivir at this dose level (1 mg/kg/day) showed 50% protection (measured by survivors/total) against the influenza virus. In the H2N2 infected control group, there were no survivors.", "In the H2N2 infected control group, there were no survivors. In view of both in vitro and in vivo data, we conclude that patchouli alcohol could be used in the treatment of human influenza virus infections. Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein.", "Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein. As the total energies and backbone root-mean-square-deviations (RMSD) in Figure 3 indicate, the energy-minimized patchouli alcohol-NA complex has been in equilibrium since about 0.5 ns, and then retains quite stable in the last 19.5 ns. It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] .", "It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] . Accordingly, the geometric and energetic analyses were made on the average structures of 0.5~20.0 ns MD trajectories, where the system has been already at equilibrium. The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ).", "The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ). As shown in Figure 4 , the patchouli alcohol was bound at the active site which also bound to oseltamivir and zanamivir [28] . As Figure 5 shows, the oxygen atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue.", "As Figure 5 shows, the oxygen atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue. The values of distances in Figure 6 further reveal that the docked complex remains rather stable throughout the simulation, with the average distances of Glu119:OE2patchouli alcohol:O and Tyr406:OH -patchouli alcohol:O less than 2.8 Å. The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) .", "The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) . Besides, patchouli alcohol was stabilized by residues Arg118, Asp151, Arg152, Trp178, Ala246, Glu276, Arg292, Asn294 and Gln347, especially residues Asp151, Arg152 and Glu276 ( Figure 5 and Table 3 ). As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] .", "As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] . The catalytic residues Asp151, Arg152 and Glu276 are crucial to the NA functions and the residues Glu119 and Tyr406 are important to stabilize the NA active sites [32, 33] . It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments.", "It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments. Patchouli alcohol matches with the NA active site and has an acceptable interaction energy. Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents.", "Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents. Patchouli alcohol and oseltamivir were obtained from Sigma Chemical Co. (St. Louis, MO, USA, purity > 99%) and was stored in glass vials with Teflon sealed caps at −20 ± 0.5 °C in the absence of light. MDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China).", "MDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China). The cells were grown in monolayer culture with Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin. The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent.", "The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent. Cells were plated out onto 96-well culture plates for cytotoxicity and anti-influenza assays, and propagated at 37 °C in an atmosphere of 5% CO 2 . The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China).", "The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China). Virus was routinely grown on MDCK cells. The stock cultures were prepared from supernatants of infected cells and stored at −80 °C. The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method.", "The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method. Briefly, cells were seeded on a microtiter plate in the absence or presence of various concentrations (20 µM -0.0098 µM) of patchouli alcohol (eight replicates) and incubated at 37 °C in a humidified atmosphere of 5% CO 2 for 72 h. The supernatants were discarded, washed with PBS twice and MTT reagent (5 mg/mL in PBS) was added to each well. After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min.", "After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min. After that the plates were read on an ELISA reader (Thermo Molecular Devices Co., Union City, USA) at 570/630 nm. The mean OD of the cell control wells was assigned a value of 100%.", "The mean OD of the cell control wells was assigned a value of 100%. The maximal non-toxic concentration (TD 0 ) and 50% cytotoxic concentration (CC 50 ) were calculated by linear regression analysis of the dose-response curves generated from the data. Inhibition of virus replication was measured by the MTT method.", "Inhibition of virus replication was measured by the MTT method. Serial dilution of the treated virus was adsorbed to the cells for 1 h at 37 °C. The residual inoculum was discared and infected cells were added with EMEM containing 2% FCS. Each assay was performed in eight replicates.", "Each assay was performed in eight replicates. After incubation for 72 h at 37 °C, the cultures were measured by MTT method as described above. The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves.", "The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves. Cells and viruses were incubated with patchouli alcohol at different stages during the viral infection cycle in order to determine the mode of antiviral action. Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells.", "Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells. Patchouli alcohol was always used at the nontoxic concentration. Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C.", "Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C. The compound was aspirated and cells were washed immediately before the influenza A (H2N2) inoculum was added. For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells.", "For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells. For analyzing the anti-influenza A (H2N2) inhibition during the adsorption period, the same amount of influenza A (H2N2) was mixed with the drug and added to the cells immediately. After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells.", "After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells. The effect of patchouli alcohol against influenza A (H2N2) was also tested during the replication period by adding it after adsorption, as typical performed in anti-influenza A (H2N2) susceptibility studies. Each assay was run in eight replicates.", "Each assay was run in eight replicates. Kunming mice, weighing 18-22 g (6 weeks of age) were purchased from Harbin Veterinary Research Institute Animal Co., Ltd. (Harbin, Heilongjiang, China) . First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline).", "First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline). The mice were orally administered with 10 mg/kg/dose patchouli alcohol, 2 mg/kg/dose patchouli alcohol or 2 mg/kg/dose oseltamivir (dissolved in 2% DMSO in physiological saline) one time daily for 7 days. The weight of mice was determined daily.", "The weight of mice was determined daily. We conducted procedures according to Principle of Laboratory Animal Care (NIH Publication No. 85 -23, revised 1985) and the guidelines of the Peking University Animal Research Committee. Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation.", "Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation. Drugs were prepared in 2% DMSO in physiological saline and administered 4 h prior to virus exposure and continued daily for 5 days. All mice were observed daily for changes in weight and for any deaths.", "All mice were observed daily for changes in weight and for any deaths. Parameters for evaluation of antiviral activity included weight loss, reduction in mortality and/or increase in mean day to death (MDD) determined through 15 days. The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] .", "The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] . For convenience, the structure is named as NA hereafter. Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 .", "Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 . The docking and molecular dynamics (MD) simulations were performed by the general protocols in the Insight II 2005 software packages, consistent with the previous literatures [24, 26, 28, 35, [37] [38] [39] . During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K).", "During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K). The MD temperature was controlled by the velocity scaling thermostat [40] . Integrations of the classical equations of motion were achieved using the Verlet algorithm.", "Integrations of the classical equations of motion were achieved using the Verlet algorithm. The systems were solvated in a large sphere of TIP3P water molecules [40] with the radius of 35.0 Å, which is enough to hold the ensembles [40] . The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals.", "The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals. The interaction energies of patchouli alcohol with NA and the respective residues at the NA active site were calculated by the Docking module [35], over the 0.5~20.0 ns MD trajectories. All results are expressed as mean values ± standard deviations (SDs) (n = 3).", "All results are expressed as mean values ± standard deviations (SDs) (n = 3). The significance of difference was calculated by one-way analysis of variance, and values p < 0.001 were considered to be significant. In conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate.", "In conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate. Our results provide the promising information for the potential use of patchouli alcohol in the treatment of influenza A (H2N2) virus infectious disease. Further mechanistic studies on the anti-influenza A virus activity are needed to support this point of view." ]

[ "Mixed viral and bacterial infections are widely described in community-acquired pneumonia; however, the clinical implications of co-infection on the associated immunopathology remain poorly studied. In this study, microRNA, mRNA and cytokine/chemokine secretion profiling were investigated for human monocyte-derived macrophages infected in-vitro with Influenza virus A/H1N1 and/or Streptococcus pneumoniae. We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions.", "We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions. We demonstrated that endogenous miRNA-200a-3p, whose expression was synergistically induced following co-infection, indirectly regulates CXCL10 expression by targeting suppressor of cytokine signaling-6 (SOCS-6), a well-known regulator of the JAK-STAT signaling pathway. Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5 years-old) hospitalized with viral and/or bacterial community-acquired pneumonia.", "Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5 years-old) hospitalized with viral and/or bacterial community-acquired pneumonia. Clinically, among the 74 cases of pneumonia, patients with identified mixed-detection had significantly higher (3.6-fold) serum IP-10 levels than those with a single detection (P = 0.03), and were significantly associated with severe pneumonia (P < 0.01). This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia.", "This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia. Text: Scientific RepoRts | 6:38532 | DOI: 10 .1038/srep38532 pathogenesis of several diseases and has been suggested as a potential biomarker of viral infection 10, 11 , late-onset bacterial infection in premature infants 12 , and a promising biomarker of sepsis and septic shock 13, 14 . Combined analysis of IP-10 and IFN-γ has also been reported as a useful biomarker for diagnosis and monitoring therapeutic efficacy in patients with active tuberculosis [15] [16] [17] , and both remain detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunction 18 .", "Combined analysis of IP-10 and IFN-γ has also been reported as a useful biomarker for diagnosis and monitoring therapeutic efficacy in patients with active tuberculosis [15] [16] [17] , and both remain detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunction 18 . With airway epithelial cells 19 , resident alveolar macrophages (AMs) and blood monocytes-derived macrophages (recruited into tissues under inflammatory conditions 20, 21 ) represent a major line of defense against both pneumococcal (through their high phagocytic capacity [22] [23] [24] ) and influenza infection 25, 26 . So far, no studies have yet focused on the intracellular mechanisms that regulate IP-10 in human blood leukocytes during mixed IAV and SP infection.", "So far, no studies have yet focused on the intracellular mechanisms that regulate IP-10 in human blood leukocytes during mixed IAV and SP infection. Several studies indicated that host non-coding small RNAs (including microRNAs) may function as immunomodulators by regulating several pivotal intracellular processes, such as the innate immune response 27 and antiviral activity 28, 29 ; both of these processes are closely related to toll-like receptor (TLR) signaling pathways. In this study, we firstly investigated the in vitro intracellular mechanisms that mediate the innate immune response in IAV and/or SP infected human monocyte-derived macrophages (MDMs).", "In this study, we firstly investigated the in vitro intracellular mechanisms that mediate the innate immune response in IAV and/or SP infected human monocyte-derived macrophages (MDMs). Using this approach, we observed that mixed-infection of MDMs induces a synergistic production of IP-10 which can be related to a miRNA-200a/JAK-STAT/SOCS-6 regulatory pathway. Subsequently, in a retrospective analysis of clinical samples collected from children ≤ 5 years-old hospitalized with pneumonia, we confirmed that serum IP-10 level could be related to both viral and/or bacterial etiologies and disease severity.", "Subsequently, in a retrospective analysis of clinical samples collected from children ≤ 5 years-old hospitalized with pneumonia, we confirmed that serum IP-10 level could be related to both viral and/or bacterial etiologies and disease severity. Characteristics of MDMs infected by IAV and/or SP. Initially, we investigated in vitro the impact of single and mixed IAV and SP infection on MDMs.", "Initially, we investigated in vitro the impact of single and mixed IAV and SP infection on MDMs. Firstly, active replication of IAV was assessed by qRT-PCR and quantification of new infectious viral particles in the cell supernatants ( Fig. 1a,b ).", "1a,b ). IAV titer increased over time after single infection with IAV and correlated with increased production of negative-strand IAV RNA. Maximum viral replication was observed at 18-24 hours post-infection, after which time both RNA replication and the quantity of infectious particles decreased.", "Maximum viral replication was observed at 18-24 hours post-infection, after which time both RNA replication and the quantity of infectious particles decreased. In this in vitro model, subsequent challenge of IAV-infected MDMs with SP had no significant impact on the production of new infectious viral particles (Fig. 1b) .", "1b) . Together, these results indicate permissive and productive infection of MDMs by IAV. Secondly, we evaluated whether MDMs are permissive for both IAV and SP infection. The presence of pneumococci within IAV-and SP-infected primary MDMs was confirmed at 8 h post-infection (Fig.", "The presence of pneumococci within IAV-and SP-infected primary MDMs was confirmed at 8 h post-infection (Fig. 1c) , suggesting that MDMs are permissive for viral and bacterial co-infection in the early steps of infection. Importantly, confocal co-detection of mixed IAV and SP was only effective following 8 h post-infection due to the bactericidal impact of SP internalization within human macrophages (after 24 h, data not shown).", "Importantly, confocal co-detection of mixed IAV and SP was only effective following 8 h post-infection due to the bactericidal impact of SP internalization within human macrophages (after 24 h, data not shown). Thirdly, we evaluated the impact of single and mixed infection with IAV and SP on MDM viability. Mixed infection significantly decreased cell viability (65.2 ± 4.5% total cell death at 48 hours post-infection; P < 0.0001) compared to single SP and IAV infection (39.6 ± 1.7% and 17.4 ± 1.1% total cell death, respectively; Fig.", "Mixed infection significantly decreased cell viability (65.2 ± 4.5% total cell death at 48 hours post-infection; P < 0.0001) compared to single SP and IAV infection (39.6 ± 1.7% and 17.4 ± 1.1% total cell death, respectively; Fig. 1d ). Taken together, these results confirmed human MDMs are permissive to mixed viral and bacterial infection.", "Taken together, these results confirmed human MDMs are permissive to mixed viral and bacterial infection. mRNA, microRNA and protein expression profiling reveal an overall induction of the host innate immune response following IAV and/or SP infection of MDMs. To investigate the innate immune response orchestrated by IAV-and SP-infected human MDMs, we firstly evaluated the expression of 84 genes involved in the innate and adaptive immune responses (Table S1) ; the major differentially-expressed genes are summarized in Fig.", "To investigate the innate immune response orchestrated by IAV-and SP-infected human MDMs, we firstly evaluated the expression of 84 genes involved in the innate and adaptive immune responses (Table S1) ; the major differentially-expressed genes are summarized in Fig. 2a . Expression profiling indicated an overall induction of genes related to the JAK-STAT, NF-Κ β and TLR signaling pathways.", "Expression profiling indicated an overall induction of genes related to the JAK-STAT, NF-Κ β and TLR signaling pathways. Indeed, all interferon-stimulated genes (ISGs) screened, including CXCL10 (fold-change [FC] = 240.9), CCL-2 (FC = 34.2) and MX-1 (FC = 151.4) were upregulated following mixed infection compared to uninfected cells, most of which are closely related to STAT-1 (FC = 52.3), IRF-7 (FC = 6.8) and IFNB1 (FC = 5.2) also found upregulated in mixed infected cells. Secondly, we investigated the endogenous microRNA expression profiles of IAV-and SP-infected MDMs.", "Secondly, we investigated the endogenous microRNA expression profiles of IAV-and SP-infected MDMs. A selection of microRNAs that were found to be differentially-expressed under different infection conditions are shown in Fig. 2b and Table S2 .", "2b and Table S2 . MiRNA-200a-3p was overexpressed after both single IAV (FC = 6.9), single SP (FC = 3.7) and mixed IAV/SP infection (FC = 7.3), indicating this miRNA may play a role in the innate immune response to viral and bacterial co-infection. Similar miRNA-200a-3p dysregulation profiles were obtained following IAV and/or SP infections of human macrophages-like (THP-1 monocytes-derived macrophages) or primary MDMs (data not shown).", "Similar miRNA-200a-3p dysregulation profiles were obtained following IAV and/or SP infections of human macrophages-like (THP-1 monocytes-derived macrophages) or primary MDMs (data not shown). Thirdly, the secreted levels of various antiviral, pro-inflammatory and immunomodulatory cytokines/chemokines were assayed in IAV-and SP-infected-THP-1 and primary MDM cell supernatants. We observed a remarkable correlation between the mRNA and protein expression profiles of single or mixed infected MDMs especially regarding CXCL-10 and IP-10 expression.", "We observed a remarkable correlation between the mRNA and protein expression profiles of single or mixed infected MDMs especially regarding CXCL-10 and IP-10 expression. Indeed, the level of IP-10 was synergistically increased in the supernatant of IAV-infected THP-1 MDMs exposed to SP (mean: 30,589 ± 16,484 pg ml −1 ) compared to single IAV infection (1,439 ± 566.5 pg ml −1 ) and single SP infection (4,472 ± 2,001 pg ml −1 ; P≤ 0.05; Fig. 2c ) at 24 hours after infection.", "2c ) at 24 hours after infection. In those cells, IP-10 expression reduced over time (48 to 72 hours), coinciding with a significant higher proportion of necrotic and apoptotic cells (Fig. 1d) . The synergistic expression of IP-10 was similarly observed at 24 hours post-infection using primary MDMs (Fig. 2d) .", "2d) . Significantly increased secretion of the other tested cytokines and chemokines was not observed post-infection, even in mixed infected MDMs (Fig. S1 ). Interestingly, a significant production of IP-10 was also observed in supernatants of primary human airway epithelial cells (HAEC) mixed-infected by IAV and SP compared to the single infections (Fig.", "Interestingly, a significant production of IP-10 was also observed in supernatants of primary human airway epithelial cells (HAEC) mixed-infected by IAV and SP compared to the single infections (Fig. 2e) . Taken together, the mRNA and protein profiling results suggested that mixed viral and bacterial infection of MDMs induces a synergistic pro-inflammatory response related to the type-1 interferon and JAK-STAT signaling pathways, with IP-10 as signature of IAV/SP co-infection.", "Taken together, the mRNA and protein profiling results suggested that mixed viral and bacterial infection of MDMs induces a synergistic pro-inflammatory response related to the type-1 interferon and JAK-STAT signaling pathways, with IP-10 as signature of IAV/SP co-infection. Among all microRNAs screened, miR-200a-3p was the most Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 overexpressed in IAV/SP co-infection of human MDMs. In the remainder of this study, we decided to investigate the interconnection between miR-200a-3p expression and the innate immune response.", "In the remainder of this study, we decided to investigate the interconnection between miR-200a-3p expression and the innate immune response. Endogenous miRNA-200a-3p expression correlates with CXCL10 (IP-10) induction following mixed IAV and SP infection of human MDMs. Using a specific Taqman probe assay targeting miR-200a-3p, we confirmed a significant upregulation of miR-200a-3p following mixed IAV and SP infection of human MDMs (Fig.", "Using a specific Taqman probe assay targeting miR-200a-3p, we confirmed a significant upregulation of miR-200a-3p following mixed IAV and SP infection of human MDMs (Fig. 3a) . In this experiment, a more marked up-regulation of miR-200a-3p was observed following IAV+ SP compared to results obtained previously (Fig. 2b) .", "2b) . This discrepancy has been attributed to the use of two different approaches to quantify miR-200a-3p expression. The use of a target-specific stem-loop reverse transcription primer in Fig. 3a allows a better sensitivity of miR-200a-3p detection compared to the non-specific fluorescent dye used in Fig. 2b .", "2b . As the general trend was suggestive of a synergistic induction of miR-200a-3p in response to mixed infection (Fig. 3a) , we hypothesized microRNA-200a-3p may play a role in the regulation of CXCL10 (IP-10), which was also synergistically upregulated in mixed-infected MDMs ( Fig.", "3a) , we hypothesized microRNA-200a-3p may play a role in the regulation of CXCL10 (IP-10), which was also synergistically upregulated in mixed-infected MDMs ( Fig. 2c and d) and primary HAEC ( Statistical analyses were performed using two-way ANOVA with Tukey's post-hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 CXCL10 (Fig.", "Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 CXCL10 (Fig. 3d) . These results suggested miR-200a-3p indirectly regulates CXCL10 and led us to hypothesize that miR-200a-3p controls a potential repressor of the JAK-STAT signaling pathway. . At 18 h after transfection, the MDMs were singly or mixed infected as described previously.", "At 18 h after transfection, the MDMs were singly or mixed infected as described previously. At 8 h post-IAV and/or SP infection, total mRNA was extracted and amplified by PCR using specific primers for the indicated genes. Values represent median ± IQR (a, c) or mean ± SEM (d, e) of three biological replicates.", "Values represent median ± IQR (a, c) or mean ± SEM (d, e) of three biological replicates. Statistical analyses were performed using a Kruskal-Wallis test (non-parametric, one-way ANOVA with Dunn's post-hoc test) for data presented in (a, c). An ordinary two-way ANOVA (with Tukey's post-hoc multiple comparison test) was used for data presented in (d, e).", "An ordinary two-way ANOVA (with Tukey's post-hoc multiple comparison test) was used for data presented in (d, e). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. MiRNA-200a-3p indirectly regulates IP-10 expression by targeting SOCS6.", "MiRNA-200a-3p indirectly regulates IP-10 expression by targeting SOCS6. As shown in Fig. 2a , several JAK-STAT signaling pathway genes were deregulated in mixed IAV-and SP-infected human MDMs; therefore, we hypothesized that miR-200a-3p directly regulates a regulator of the JAK-STAT signaling pathway.", "2a , several JAK-STAT signaling pathway genes were deregulated in mixed IAV-and SP-infected human MDMs; therefore, we hypothesized that miR-200a-3p directly regulates a regulator of the JAK-STAT signaling pathway. Predictive target analysis indicated that the 3' UTR of suppressor of cytokine signaling-6 (SOCS6) may be targeted by miR-200a-3p (Fig. 3b) .", "3b) . SOCS proteins constitute a class of negative regulators of JAK-STAT signaling pathways that are induced by both cytokines and TLR signaling. MiRNA-200a-3p was not predicted to target any of the other six members of the SOCS gene family.", "MiRNA-200a-3p was not predicted to target any of the other six members of the SOCS gene family. Transfection of human MDMs with MIM-200a downregulated SOCS6 (FC = 0.57) while inhibition of miR-200a-3p (INH-200a) upregulated SOCS6 (FC = 1.55), confirming that miR-200a-3p effectively regulates the expression of SOCS6 (Fig. 3e) .", "3e) . Moreover, SOCS6 was synergistically downregulated in IAV-or IAV/SP-infected MDMs overexpressing miRNA-200a (Fig. 3e) , suggesting that both infection and miR-200a-3p negatively regulate the expression of SOCS6. Finally, western blotting confirmed that expression of SOCS-6 sharply reduced following infection, especially after mixed IAV and SP infection (Fig. 3f) .", "3f) . These results indicate miR-200a-3p is strongly induced in response to mixed viral and bacterial co-infection, which in turn leads to downregulation of the JAK-STAT regulator SOCS-6 at both the mRNA and protein levels and subsequent upregulation of IP-10. analyses demonstrated mixed IAV and SP infection of human MDMs and HAEC induced significant production of IP-10.", "analyses demonstrated mixed IAV and SP infection of human MDMs and HAEC induced significant production of IP-10. As blood leukocytes and respiratory tract epithelial cells actively contribute to inflammation during pneumonia, we hypothesized the level of IP-10 in serum of patient with pneumonia may be both indicative of mixed respiratory infection and disease severity. As part of a prospective, hospital-based, multicenter case-control study on the etiology of pneumonia among children under 5-years-old, a total of 74 patients (44 male, 30 female) were included in this pilot evaluation.", "As part of a prospective, hospital-based, multicenter case-control study on the etiology of pneumonia among children under 5-years-old, a total of 74 patients (44 male, 30 female) were included in this pilot evaluation. According to WHO guidelines, retrospective analysis indicated 44 (59.5%) children had clinical signs of non-severe pneumonia and 30 (40.5%) children had signs of severe pneumonia. The main patient characteristics at inclusion are shown in Table 1 .", "The main patient characteristics at inclusion are shown in Table 1 . Patients with severe pneumonia had significant more recorded episodes of dyspnea (P < 0.001), cyanosis (P = 0.03), lower chest indrawing (P < 0.001), dullness to percussion (P < 0.001) and lethargy (P < 0.001) during chest examination than patient with non-severe pneumonia. Moreover, pleural effusions were significantly more observed among critically ill patients and the duration of hospitalization was significantly longer for the children with severe pneumonia than for those with non-severe pneumonia (P = 0.0015).", "Moreover, pleural effusions were significantly more observed among critically ill patients and the duration of hospitalization was significantly longer for the children with severe pneumonia than for those with non-severe pneumonia (P = 0.0015). Two deaths occurred within the group of children retrospectively defined with severe pneumonia. Evaluation of the systemic inflammatory response of the 74 cases is shown in Table 2 .", "Evaluation of the systemic inflammatory response of the 74 cases is shown in Table 2 . Serum level of CRP, IP-10, PCT, G-CSF, IL-6, IL-8 and MIP-1β were significantly more elevated in serum samples from critically ill patients. Patients with severe pneumonia had significantly higher (4.2-fold) serum IP-10 levels than those with a non-severe pneumonia (P < 0.001) suggesting IP-10 as a promising prognostic marker in pneumonia.", "Patients with severe pneumonia had significantly higher (4.2-fold) serum IP-10 levels than those with a non-severe pneumonia (P < 0.001) suggesting IP-10 as a promising prognostic marker in pneumonia. Diagnostic accuracy measures for predicting pneumonia severity using blood-based biomarkers are summarized in Table S3 . Briefly, in this study, the optimal IP-10 cut-off value for identifying patient with severe pneumonia was 4,240 pg ml −1 , with an area under the receiver operating characteristic curve of 0.69 (95% CI, 0.57 to 0.82, P < 0.001).", "Briefly, in this study, the optimal IP-10 cut-off value for identifying patient with severe pneumonia was 4,240 pg ml −1 , with an area under the receiver operating characteristic curve of 0.69 (95% CI, 0.57 to 0.82, P < 0.001). Defining as positive a serum IP-10 level above this cut-off resulted in a sensitivity of 63.3%, specificity of 63.6% and a positive likelihood ratio of 1.74. Prognostic values of IP-10 were closed to procalcitonin (PCT; AUC = 0.70; 95% IC, 0.58 to 0.82, P < 0.001) and IL-6 (AUC = 0.70; 95% IC, 0.58-0.83, P < 0.001).", "Prognostic values of IP-10 were closed to procalcitonin (PCT; AUC = 0.70; 95% IC, 0.58 to 0.82, P < 0.001) and IL-6 (AUC = 0.70; 95% IC, 0.58-0.83, P < 0.001). Multiplex PCR-based screening of respiratory and blood samples reveal a high variety of pathogen associations (Table 3) . Respiratory viruses were detected in the nasal aspirates (NAs) of 63/74 patients (85.1%).", "Respiratory viruses were detected in the nasal aspirates (NAs) of 63/74 patients (85.1%). Etiological bacteria of pneumonia (S. pneumoniae, n = 19; S. aureus, n = 1; or H. influenzae type B, n = 7) were identified via real-time PCR in the blood samples of 27/74 (36.5%) of the patients. Multiplex PCR assays allowed the identification of respiratory bacteria in the blood of 19 patients with negative blood culture results.", "Multiplex PCR assays allowed the identification of respiratory bacteria in the blood of 19 patients with negative blood culture results. Among the 74 cases PCR-positive for respiratory pathogens, a single virus or bacteria were detected in the NAs of 7 (9.4%) and 3 (4.0%) patients, respectively; these 10/74 (13.5%) cases were defined as the single infection group. The mixed infection group included the 62/74 (83.8%) cases in which (1) multiple viruses and/or bacteria were identified in NAs (38/74; 51.3%) without any bacteria identified in blood samples or (2) one or more viruses and/or bacteria were identified in NAs and associated with a blood bacteremia (24/74; 32.4%).", "The mixed infection group included the 62/74 (83.8%) cases in which (1) multiple viruses and/or bacteria were identified in NAs (38/74; 51.3%) without any bacteria identified in blood samples or (2) one or more viruses and/or bacteria were identified in NAs and associated with a blood bacteremia (24/74; 32.4%). We evaluated whether IP-10 serum level could correlate with the viral and bacterial etiologies of pneumonia. Patients with mixed infection had significant higher (3.6-fold) IP-10 serum level than patient with single detection (P = 0.03; Table 4 ).", "Patients with mixed infection had significant higher (3.6-fold) IP-10 serum level than patient with single detection (P = 0.03; Table 4 ). A stratified analysis reveals that the highest IP-10 serum level was observed among patients with both several respiratory pathogens identified (mixed-detection group) and severe pneumonia (14,427 pg ml −1 , IQR (3,981-82,994). In detail, a remarkable IP-10 serum level (142,531 pg ml −1 ), representing 33-fold higher above cut-off value predicting pneumonia severity was observed in patient with hRV in NA co-detected with S. pneumoniae (serotype 14) in pleural effusion and blood.", "In detail, a remarkable IP-10 serum level (142,531 pg ml −1 ), representing 33-fold higher above cut-off value predicting pneumonia severity was observed in patient with hRV in NA co-detected with S. pneumoniae (serotype 14) in pleural effusion and blood. In concordance with our in-vitro model of co-infection, a significant IP-10 level (90,338 pg ml −1 ) was quantified in blood sample of patient with severe bacteremic pneumococcal (serotype 14) pneumonia with a positive co-detection of Influenza B virus in NA. Taken together, these results suggest that high serum IP-10 levels are significantly associated with mixed viral and bacterial detection and also related to pneumonia pathogenesis.", "Taken together, these results suggest that high serum IP-10 levels are significantly associated with mixed viral and bacterial detection and also related to pneumonia pathogenesis. This study provides additional in vitro and clinical data to improve our understanding of the immunopathology of mixed viral and bacterial pneumonia (Fig. 4) .", "4) . The in vitro model of influenza and pneumococcal superinfection of human MDMs demonstrated that mixed infection synergistically induced release of the pro-inflammatory chemokine IP-10, strongly suggesting human Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 blood leukocytes contribute to the immunopathology of pneumonia. Additionally, transcriptomics and omics analyses provided new data on the inflammatory pathways that are activated during mixed infection and related to synergistic induction of the pro-inflammatory chemokine IP-10 in mixed infected cells.", "Additionally, transcriptomics and omics analyses provided new data on the inflammatory pathways that are activated during mixed infection and related to synergistic induction of the pro-inflammatory chemokine IP-10 in mixed infected cells. Our observations are consistent with a recent study describing IP-10 induction as host-proteome signature of both viral and bacterial infections 30 . Of the differentially-expressed genes observed in mixed infected MDMs, the transcription factors STAT-1 and IRF-7 appear to play crucial roles in the regulation of interferon-stimulated genes including CXCL10 (IP-10).", "Of the differentially-expressed genes observed in mixed infected MDMs, the transcription factors STAT-1 and IRF-7 appear to play crucial roles in the regulation of interferon-stimulated genes including CXCL10 (IP-10). By focusing on the intracellular mechanisms that regulate inflammatory pathways, we demonstrated a novel role for miRNA-200a-3p in the regulation of CXCL10 (IP-10). These observations are consistent with previous reports showing that RNA virus infection upregulates miR-155 in macrophages and dendritic cells and also regulates suppressor of cytokine signaling 1 (SOCS1), suggesting the existence of a miRNA/JAK-STAT/SOCS regulatory pathway during viral infection 29 .", "These observations are consistent with previous reports showing that RNA virus infection upregulates miR-155 in macrophages and dendritic cells and also regulates suppressor of cytokine signaling 1 (SOCS1), suggesting the existence of a miRNA/JAK-STAT/SOCS regulatory pathway during viral infection 29 . Our study suggests co-infection leads to overexpression of miR-200a-3p, which in turn targets and downregulates the JAK-STAT regulator SOCS-6 and consequently increases CXCL10 (IP-10) expression. Interestingly, a complementary in-silico approach reveals that several microRNAs that were found dysregulated in our experiments of IAV and SP co-infection of MDMs or HAEC, might target several genes of SOCS family and play similar role than miR-200a-3p.", "Interestingly, a complementary in-silico approach reveals that several microRNAs that were found dysregulated in our experiments of IAV and SP co-infection of MDMs or HAEC, might target several genes of SOCS family and play similar role than miR-200a-3p. Indeed, miRNA-142-3p might target SOCS4, 5, 6 mRNA while miRNA-194-5p might target SOCS2, 3, 4, 5 and 7 mRNA. These observations underline that intra-cellular regulation of IP-10 is not limited to the contribution of a sole microRNA.", "These observations underline that intra-cellular regulation of IP-10 is not limited to the contribution of a sole microRNA. A complex inter-relationship between numerous host microRNAs and inhibitors of the JAK-STAT signaling pathway occur to control host innate inflammatory response against viral and/or bacterial infections. Clinically, the majority of pediatric CAP cases in this study were associated with both positive viral and/or bacterial detection.", "Clinically, the majority of pediatric CAP cases in this study were associated with both positive viral and/or bacterial detection. Respiratory microorganisms were detected in 97% of cases; 51.3% of which were viral-viral, viral-bacterial or bacterial-bacterial co-detected only in nasal aspirates, 32.4% of which co-detected in both nasal aspirates and blood samples. These data are consistent with previous etiological studies of pediatric CAP 3,31-33 .", "These data are consistent with previous etiological studies of pediatric CAP 3,31-33 . S. pneumoniae was the major bacteria identified in blood (19/74; 25.7%) and mainly co-detected with respiratory viruses in NAs (16/19; 84.2%). We observed a very high diversity of viral and bacterial associations in biological samples from children with pneumonia.", "We observed a very high diversity of viral and bacterial associations in biological samples from children with pneumonia. In comparison with IAV and SP14 combination evaluated in-vitro, no pneumonia cases were singly influenza and pneumococcus infected, and no similar co-detection with those two pathogens has been clinically observed. Nevertheless, Influenza B (IVB) virus was identified in 5 patients and two of them had a positive SP co-detection in blood (one non-typable strain and one serotype 14 using our molecular typing test).", "Nevertheless, Influenza B (IVB) virus was identified in 5 patients and two of them had a positive SP co-detection in blood (one non-typable strain and one serotype 14 using our molecular typing test). IVB and SP14 combination seems to be the nearest pathogen co-detection to that in-vitro investigated. Clinically, this co-detection was associated with both a very high IP-10 expression and a very severe pneumonia case definition.", "Clinically, this co-detection was associated with both a very high IP-10 expression and a very severe pneumonia case definition. Interestingly, our translational pilot evaluation reveals IP-10 expression can be induced by several different viral and/or bacterial combinations. As immune response to each pathogen is different, further in-vitro investigations using different pathogens associations are needed to better characterize the mechanisms involved in the immunopathology of pneumonia.", "As immune response to each pathogen is different, further in-vitro investigations using different pathogens associations are needed to better characterize the mechanisms involved in the immunopathology of pneumonia. In this cohort, highest serum IP-10 levels were identified among patients with both several pathogen detected and severe pneumonia, suggesting a significant role of IP-10 on pneumonia pathogenesis. Indeed, high plasma levels of IP-10 have previously been reported in patients with sepsis 12 , and were associated with high mortality rate, especially among patients with CAP 34 .", "Indeed, high plasma levels of IP-10 have previously been reported in patients with sepsis 12 , and were associated with high mortality rate, especially among patients with CAP 34 . Additionally, the IP-10-CXCR3 axis has been related to acute immune lung injury and lymphocyte apoptosis during the development of severe acute respiratory syndrome (SARS) 35, 36 . Moreover, an in vivo study that modeled influenza and pneumococcal superinfection in mice indicated that pro-inflammatory chemokines, including IP-10, play a crucial role in influenza-induced susceptibility to lung neutrophilia, severe immunopathology and mortality 37 .", "Moreover, an in vivo study that modeled influenza and pneumococcal superinfection in mice indicated that pro-inflammatory chemokines, including IP-10, play a crucial role in influenza-induced susceptibility to lung neutrophilia, severe immunopathology and mortality 37 . In this study, markedly elevated IP-10 (92,809 pg ml −1 ) combined with the highest PCT level (74.4 pg ml −1 ) were quantified in the serum sample of a child who died, in whom S. pneumoniae (serotype 9 V) was identified in the blood (PCR and blood culture) and co-detected with Haemophilus influenzae type B in nasal aspirate. These observations suggest an interrelationship between co-detection, elevated serum IP-10 and the pathogenesis of pneumonia.", "These observations suggest an interrelationship between co-detection, elevated serum IP-10 and the pathogenesis of pneumonia. Several limitations of this pilot translational study need to be acknowledged before concluding mixed infection is related to elevated IP-10 and disease severity. Indeed, although viral shedding (e.g., of HRV and HBoV) is common in asymptomatic children, we were unable to evaluate the levels of immunomodulators in the serum samples of a control group.", "Indeed, although viral shedding (e.g., of HRV and HBoV) is common in asymptomatic children, we were unable to evaluate the levels of immunomodulators in the serum samples of a control group. Moreover, although the samples were collected within the first 24 hours after admission, only a single blood sample was processed for each patient. Therefore, a larger, longitudinal study on the etiology and severity of pneumonia will be necessary to confirm these results.", "Therefore, a larger, longitudinal study on the etiology and severity of pneumonia will be necessary to confirm these results. In conclusion, the present findings suggest that mixed respiratory infections and IP-10 may play major, interconnected roles in the pathogenesis of pneumonia. Clinically, assessment and monitoring of induced IP-10 serum level may assist clinicians to improve diagnosis and patient management of severe community-acquired pneumonia.", "Clinically, assessment and monitoring of induced IP-10 serum level may assist clinicians to improve diagnosis and patient management of severe community-acquired pneumonia. Viral and bacterial strains. The 10 ng ml −1 M-CSF (Miltenyi Biotec).", "Viral and bacterial strains. The 10 ng ml −1 M-CSF (Miltenyi Biotec). THP− 1 MDMs were obtained by culturing cells with 10 ng ml -1 phorbol myristate acetate (PMA; Invivogen, Toulouse, France) for 72 hours.", "THP− 1 MDMs were obtained by culturing cells with 10 ng ml -1 phorbol myristate acetate (PMA; Invivogen, Toulouse, France) for 72 hours. Human airway epithelial cells (HAEC, bronchial cell type) originated from a 54-years old woman with no pathology reported (batch number MD056501) were provided by Mucilair (Epithelix, Geneva, Switzerland). Sterility, tissue integrity (TEER), mucus production and cilia beating frequency have been certified by the company.", "Sterility, tissue integrity (TEER), mucus production and cilia beating frequency have been certified by the company. Gene expression profiling. Total cellular mRNA was purified using the RNeasy kit (Qiagen, Hilden, Germany). Reverse-transcription of total mRNA was performed using the RT 2 First Strand Kit (SABiosciences, Hilden, Germany).", "Reverse-transcription of total mRNA was performed using the RT 2 First Strand Kit (SABiosciences, Hilden, Germany). The expression of 84 genes involved in the human innate and adaptive immune responses was evaluated using the RT 2 profiler ™ PCR Array (SABiosciences) according to the manufacturer's recommendations. The Δ Δ Ct method was applied to calculate the fold changes in gene expression for each gene relative to uninfected control cells using the web-based RT 2 profiler PCR Array Data Analysis software (SABiosciences).", "The Δ Δ Ct method was applied to calculate the fold changes in gene expression for each gene relative to uninfected control cells using the web-based RT 2 profiler PCR Array Data Analysis software (SABiosciences). MicroRNA profiling array. Total cellular microRNAs were purified using the miRNeasy Mini kit (Qiagen) and reverse-transcribed using the miScript Reverse Transcription kit (Qiagen).", "Total cellular microRNAs were purified using the miRNeasy Mini kit (Qiagen) and reverse-transcribed using the miScript Reverse Transcription kit (Qiagen). The profiling of 84 miRNAs was performed using the Human Immunopathology miScript miRNA PCR Array kit (Qiagen) according to the manufacturer's instructions. Data were analyzed using the miScript miRNA PCR array data analysis web portal.", "Data were analyzed using the miScript miRNA PCR array data analysis web portal. In silico miRNA target prediction. MiRNA target genes were retrieved and compiled using TargetScan 38 and microRNA.org resource 39 . The interactions between miRNAs and intracellular pathways were predicted using DIANA-miRPath v2.0 40 .", "The interactions between miRNAs and intracellular pathways were predicted using DIANA-miRPath v2.0 40 . THP-1 MDMs were seeded in 24-well plates (0.5 × 10 6 per well) in triplicate, exposed to Influenza A H1N1 (A/Solomon islands/3/2006) virus (IAV) under serum-free conditions for 1 hour and then cultured for 3 hours in fresh RPMI-1640 containing 2% FBS. Streptococcus pneumoniae (SP) serotype 14 was added at 4 hours after IAV infection.", "Streptococcus pneumoniae (SP) serotype 14 was added at 4 hours after IAV infection. Gentamicin (10 μ g ml −1 ) was added 2 hours after SP infection (i.e. 6 hours post-influenza infection) and maintained in the culture media throughout the experiment to kill extracellular bacteria and limit bacterial growth.", "6 hours post-influenza infection) and maintained in the culture media throughout the experiment to kill extracellular bacteria and limit bacterial growth. Cell viability was determined by flow-cytometry using the FITC/Annexin V apoptosis detection kit (BD Biosciences), according to the manufacturer's instructions. #4427975) .", "#4427975) . In this assay, fold changes have been defined by the Δ Δ Ct method using control RNU-44 and -48 as reference microRNAs. Total mRNA was purified from transfected and infected MDMs using the RNeasy kit (Qiagen) and specific primers were used to amplify transforming growth factor beta-2 (TGFB2; F: 5′ -CCATCCCGCCCACTTTCTAC-3′ , R: 5′ -AGCTCAATCCGTTGTTCAGGC-3′ ), SOCS6 (F: 5′ -AAGAATTCATCCCTTGGATTAGGTAAC-3′ , R: 5′ -CAGACTGGAGGTCGTGGAA-3′ ) 41 43 , and 3) absence of wheezing at auscultation, and, 4) first symptoms appearing within the last 14 days, and 5) radiological confirmation of pneumonia as per WHO guidelines 44 .", "Total mRNA was purified from transfected and infected MDMs using the RNeasy kit (Qiagen) and specific primers were used to amplify transforming growth factor beta-2 (TGFB2; F: 5′ -CCATCCCGCCCACTTTCTAC-3′ , R: 5′ -AGCTCAATCCGTTGTTCAGGC-3′ ), SOCS6 (F: 5′ -AAGAATTCATCCCTTGGATTAGGTAAC-3′ , R: 5′ -CAGACTGGAGGTCGTGGAA-3′ ) 41 43 , and 3) absence of wheezing at auscultation, and, 4) first symptoms appearing within the last 14 days, and 5) radiological confirmation of pneumonia as per WHO guidelines 44 . Based on these primary criteria defining pneumonia cases, all 74 cases were retrospectively re-evaluated according to the WHO \"Pocket book of hospital care for children\" 45 criteria to evaluate pneumonia severity. Cases that died during the study, or who had at least one additional clinical signs including central cyanosis, dullness to percussion during chest examination, prostration/lethargy, pleural effusion observed on chest radiography were retrospectively included in the severe pneumonia group.", "Cases that died during the study, or who had at least one additional clinical signs including central cyanosis, dullness to percussion during chest examination, prostration/lethargy, pleural effusion observed on chest radiography were retrospectively included in the severe pneumonia group. Patients without any of these additional clinical signs were included in the non-severe pneumonia group. Table 4 .", "Table 4 . a IP-10 values are expressed in pg ml -1 . IP-10 concentration differences between groups were compared using unpaired Mann-Whitney tests; significant changes (P < 0.05) are in bold. Clinical and molecular analysis. Nasopharyngeal aspirates (NAs) and whole blood samples were collected from children within 24 hours of admission.", "Nasopharyngeal aspirates (NAs) and whole blood samples were collected from children within 24 hours of admission. Whole blood samples were used for complete blood counts, blood culture and multiplex real-time PCR to identify Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae type B 46 . S. pneumoniae serotypes were defined using a 11 multiplex real-time PCR assay targeting the 40 most frequently represented serotypes or serogroups according to protocol developed by Messaoudi et al.", "S. pneumoniae serotypes were defined using a 11 multiplex real-time PCR assay targeting the 40 most frequently represented serotypes or serogroups according to protocol developed by Messaoudi et al. 47 . Serum C-reactive protein (CRP; AssayPro, St. Charles, Missouri, United States) and Procalcitonin (PCT; VIDAS B.R.A.H.M.S; bioMérieux) were quantified from whole-blood samples.", "Serum C-reactive protein (CRP; AssayPro, St. Charles, Missouri, United States) and Procalcitonin (PCT; VIDAS B.R.A.H.M.S; bioMérieux) were quantified from whole-blood samples. Multiplex real-time non quantitative PCR (Fast-Track Diagnostic, Sliema, Malta) was used to detect 19 viruses and five bacteria in the respiratory specimens (NAs and pleural effusions). Mixed detection was defined as 1) PCR-positive for multiple viruses in NAs, 2) positive blood culture or PCR-positive for multiple bacteria in blood or 3) PCR-positive for one or multiple viruses in NAs and one or multiple bacteria in blood (identified by PCR and blood culture).", "Mixed detection was defined as 1) PCR-positive for multiple viruses in NAs, 2) positive blood culture or PCR-positive for multiple bacteria in blood or 3) PCR-positive for one or multiple viruses in NAs and one or multiple bacteria in blood (identified by PCR and blood culture). Ethical approval. The study protocol, informed consent statement, clinical research form, any amendments and all other study documents were submitted to and approved by the Ethical Committee of the Instituto de Investigaciones en Ciencias de la Salud, the Universidad Nacional de Asunción (IICS-UNA) and the Hospital Pediátrico Niños de Acosta Ñu.", "The study protocol, informed consent statement, clinical research form, any amendments and all other study documents were submitted to and approved by the Ethical Committee of the Instituto de Investigaciones en Ciencias de la Salud, the Universidad Nacional de Asunción (IICS-UNA) and the Hospital Pediátrico Niños de Acosta Ñu. Informed consent was obtained from all subjects involved in this study. The clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki.", "The clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki. Statistical analysis. The Chi-square test and Fisher's exact test were used to compare categorical variables; continuous variables and non-normally distributed data were compared using the Mann-Whitney U-test; normally distributed data were compared using unpaired t-tests.", "The Chi-square test and Fisher's exact test were used to compare categorical variables; continuous variables and non-normally distributed data were compared using the Mann-Whitney U-test; normally distributed data were compared using unpaired t-tests. Comparative analyses between experimental conditions (i.e., MOCK, IAV, SP or IAV + SP) were performed using one-way ANOVA with Tukey's post-hoc test or Kruskal-Wallis analysis with Dunn's post-hoc tests. Receiver operating curve (ROC) analysis was used to determine the optimal cut-off value for IP-10 to differentiate between non-severe and severe pneumonia cases.", "Receiver operating curve (ROC) analysis was used to determine the optimal cut-off value for IP-10 to differentiate between non-severe and severe pneumonia cases. P < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism (La Jolla, California, United States)." ]

[ "Mixed viral and bacterial infections are widely described in community-acquired pneumonia; however, the clinical implications of co-infection on the associated immunopathology remain poorly studied. In this study, microRNA, mRNA and cytokine/chemokine secretion profiling were investigated for human monocyte-derived macrophages infected in-vitro with Influenza virus A/H1N1 and/or Streptococcus pneumoniae. We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions.", "We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions. We demonstrated that endogenous miRNA-200a-3p, whose expression was synergistically induced following co-infection, indirectly regulates CXCL10 expression by targeting suppressor of cytokine signaling-6 (SOCS-6), a well-known regulator of the JAK-STAT signaling pathway. Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5 years-old) hospitalized with viral and/or bacterial community-acquired pneumonia.", "Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5 years-old) hospitalized with viral and/or bacterial community-acquired pneumonia. Clinically, among the 74 cases of pneumonia, patients with identified mixed-detection had significantly higher (3.6-fold) serum IP-10 levels than those with a single detection (P = 0.03), and were significantly associated with severe pneumonia (P < 0.01). This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia.", "This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia. Text: Scientific RepoRts | 6:38532 | DOI: 10 .1038/srep38532 pathogenesis of several diseases and has been suggested as a potential biomarker of viral infection 10, 11 , late-onset bacterial infection in premature infants 12 , and a promising biomarker of sepsis and septic shock 13, 14 . Combined analysis of IP-10 and IFN-γ has also been reported as a useful biomarker for diagnosis and monitoring therapeutic efficacy in patients with active tuberculosis [15] [16] [17] , and both remain detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunction 18 .", "Combined analysis of IP-10 and IFN-γ has also been reported as a useful biomarker for diagnosis and monitoring therapeutic efficacy in patients with active tuberculosis [15] [16] [17] , and both remain detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunction 18 . With airway epithelial cells 19 , resident alveolar macrophages (AMs) and blood monocytes-derived macrophages (recruited into tissues under inflammatory conditions 20, 21 ) represent a major line of defense against both pneumococcal (through their high phagocytic capacity [22] [23] [24] ) and influenza infection 25, 26 . So far, no studies have yet focused on the intracellular mechanisms that regulate IP-10 in human blood leukocytes during mixed IAV and SP infection.", "So far, no studies have yet focused on the intracellular mechanisms that regulate IP-10 in human blood leukocytes during mixed IAV and SP infection. Several studies indicated that host non-coding small RNAs (including microRNAs) may function as immunomodulators by regulating several pivotal intracellular processes, such as the innate immune response 27 and antiviral activity 28, 29 ; both of these processes are closely related to toll-like receptor (TLR) signaling pathways. In this study, we firstly investigated the in vitro intracellular mechanisms that mediate the innate immune response in IAV and/or SP infected human monocyte-derived macrophages (MDMs).", "In this study, we firstly investigated the in vitro intracellular mechanisms that mediate the innate immune response in IAV and/or SP infected human monocyte-derived macrophages (MDMs). Using this approach, we observed that mixed-infection of MDMs induces a synergistic production of IP-10 which can be related to a miRNA-200a/JAK-STAT/SOCS-6 regulatory pathway. Subsequently, in a retrospective analysis of clinical samples collected from children ≤ 5 years-old hospitalized with pneumonia, we confirmed that serum IP-10 level could be related to both viral and/or bacterial etiologies and disease severity.", "Subsequently, in a retrospective analysis of clinical samples collected from children ≤ 5 years-old hospitalized with pneumonia, we confirmed that serum IP-10 level could be related to both viral and/or bacterial etiologies and disease severity. Characteristics of MDMs infected by IAV and/or SP. Initially, we investigated in vitro the impact of single and mixed IAV and SP infection on MDMs.", "Initially, we investigated in vitro the impact of single and mixed IAV and SP infection on MDMs. Firstly, active replication of IAV was assessed by qRT-PCR and quantification of new infectious viral particles in the cell supernatants ( Fig. 1a,b ).", "1a,b ). IAV titer increased over time after single infection with IAV and correlated with increased production of negative-strand IAV RNA. Maximum viral replication was observed at 18-24 hours post-infection, after which time both RNA replication and the quantity of infectious particles decreased.", "Maximum viral replication was observed at 18-24 hours post-infection, after which time both RNA replication and the quantity of infectious particles decreased. In this in vitro model, subsequent challenge of IAV-infected MDMs with SP had no significant impact on the production of new infectious viral particles (Fig. 1b) .", "1b) . Together, these results indicate permissive and productive infection of MDMs by IAV. Secondly, we evaluated whether MDMs are permissive for both IAV and SP infection. The presence of pneumococci within IAV-and SP-infected primary MDMs was confirmed at 8 h post-infection (Fig.", "The presence of pneumococci within IAV-and SP-infected primary MDMs was confirmed at 8 h post-infection (Fig. 1c) , suggesting that MDMs are permissive for viral and bacterial co-infection in the early steps of infection. Importantly, confocal co-detection of mixed IAV and SP was only effective following 8 h post-infection due to the bactericidal impact of SP internalization within human macrophages (after 24 h, data not shown).", "Importantly, confocal co-detection of mixed IAV and SP was only effective following 8 h post-infection due to the bactericidal impact of SP internalization within human macrophages (after 24 h, data not shown). Thirdly, we evaluated the impact of single and mixed infection with IAV and SP on MDM viability. Mixed infection significantly decreased cell viability (65.2 ± 4.5% total cell death at 48 hours post-infection; P < 0.0001) compared to single SP and IAV infection (39.6 ± 1.7% and 17.4 ± 1.1% total cell death, respectively; Fig.", "Mixed infection significantly decreased cell viability (65.2 ± 4.5% total cell death at 48 hours post-infection; P < 0.0001) compared to single SP and IAV infection (39.6 ± 1.7% and 17.4 ± 1.1% total cell death, respectively; Fig. 1d ). Taken together, these results confirmed human MDMs are permissive to mixed viral and bacterial infection.", "Taken together, these results confirmed human MDMs are permissive to mixed viral and bacterial infection. mRNA, microRNA and protein expression profiling reveal an overall induction of the host innate immune response following IAV and/or SP infection of MDMs. To investigate the innate immune response orchestrated by IAV-and SP-infected human MDMs, we firstly evaluated the expression of 84 genes involved in the innate and adaptive immune responses (Table S1) ; the major differentially-expressed genes are summarized in Fig.", "To investigate the innate immune response orchestrated by IAV-and SP-infected human MDMs, we firstly evaluated the expression of 84 genes involved in the innate and adaptive immune responses (Table S1) ; the major differentially-expressed genes are summarized in Fig. 2a . Expression profiling indicated an overall induction of genes related to the JAK-STAT, NF-Κ β and TLR signaling pathways.", "Expression profiling indicated an overall induction of genes related to the JAK-STAT, NF-Κ β and TLR signaling pathways. Indeed, all interferon-stimulated genes (ISGs) screened, including CXCL10 (fold-change [FC] = 240.9), CCL-2 (FC = 34.2) and MX-1 (FC = 151.4) were upregulated following mixed infection compared to uninfected cells, most of which are closely related to STAT-1 (FC = 52.3), IRF-7 (FC = 6.8) and IFNB1 (FC = 5.2) also found upregulated in mixed infected cells. Secondly, we investigated the endogenous microRNA expression profiles of IAV-and SP-infected MDMs.", "Secondly, we investigated the endogenous microRNA expression profiles of IAV-and SP-infected MDMs. A selection of microRNAs that were found to be differentially-expressed under different infection conditions are shown in Fig. 2b and Table S2 .", "2b and Table S2 . MiRNA-200a-3p was overexpressed after both single IAV (FC = 6.9), single SP (FC = 3.7) and mixed IAV/SP infection (FC = 7.3), indicating this miRNA may play a role in the innate immune response to viral and bacterial co-infection. Similar miRNA-200a-3p dysregulation profiles were obtained following IAV and/or SP infections of human macrophages-like (THP-1 monocytes-derived macrophages) or primary MDMs (data not shown).", "Similar miRNA-200a-3p dysregulation profiles were obtained following IAV and/or SP infections of human macrophages-like (THP-1 monocytes-derived macrophages) or primary MDMs (data not shown). Thirdly, the secreted levels of various antiviral, pro-inflammatory and immunomodulatory cytokines/chemokines were assayed in IAV-and SP-infected-THP-1 and primary MDM cell supernatants. We observed a remarkable correlation between the mRNA and protein expression profiles of single or mixed infected MDMs especially regarding CXCL-10 and IP-10 expression.", "We observed a remarkable correlation between the mRNA and protein expression profiles of single or mixed infected MDMs especially regarding CXCL-10 and IP-10 expression. Indeed, the level of IP-10 was synergistically increased in the supernatant of IAV-infected THP-1 MDMs exposed to SP (mean: 30,589 ± 16,484 pg ml −1 ) compared to single IAV infection (1,439 ± 566.5 pg ml −1 ) and single SP infection (4,472 ± 2,001 pg ml −1 ; P≤ 0.05; Fig. 2c ) at 24 hours after infection.", "2c ) at 24 hours after infection. In those cells, IP-10 expression reduced over time (48 to 72 hours), coinciding with a significant higher proportion of necrotic and apoptotic cells (Fig. 1d) . The synergistic expression of IP-10 was similarly observed at 24 hours post-infection using primary MDMs (Fig. 2d) .", "2d) . Significantly increased secretion of the other tested cytokines and chemokines was not observed post-infection, even in mixed infected MDMs (Fig. S1 ). Interestingly, a significant production of IP-10 was also observed in supernatants of primary human airway epithelial cells (HAEC) mixed-infected by IAV and SP compared to the single infections (Fig.", "Interestingly, a significant production of IP-10 was also observed in supernatants of primary human airway epithelial cells (HAEC) mixed-infected by IAV and SP compared to the single infections (Fig. 2e) . Taken together, the mRNA and protein profiling results suggested that mixed viral and bacterial infection of MDMs induces a synergistic pro-inflammatory response related to the type-1 interferon and JAK-STAT signaling pathways, with IP-10 as signature of IAV/SP co-infection.", "Taken together, the mRNA and protein profiling results suggested that mixed viral and bacterial infection of MDMs induces a synergistic pro-inflammatory response related to the type-1 interferon and JAK-STAT signaling pathways, with IP-10 as signature of IAV/SP co-infection. Among all microRNAs screened, miR-200a-3p was the most Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 overexpressed in IAV/SP co-infection of human MDMs. In the remainder of this study, we decided to investigate the interconnection between miR-200a-3p expression and the innate immune response.", "In the remainder of this study, we decided to investigate the interconnection between miR-200a-3p expression and the innate immune response. Endogenous miRNA-200a-3p expression correlates with CXCL10 (IP-10) induction following mixed IAV and SP infection of human MDMs. Using a specific Taqman probe assay targeting miR-200a-3p, we confirmed a significant upregulation of miR-200a-3p following mixed IAV and SP infection of human MDMs (Fig.", "Using a specific Taqman probe assay targeting miR-200a-3p, we confirmed a significant upregulation of miR-200a-3p following mixed IAV and SP infection of human MDMs (Fig. 3a) . In this experiment, a more marked up-regulation of miR-200a-3p was observed following IAV+ SP compared to results obtained previously (Fig. 2b) .", "2b) . This discrepancy has been attributed to the use of two different approaches to quantify miR-200a-3p expression. The use of a target-specific stem-loop reverse transcription primer in Fig. 3a allows a better sensitivity of miR-200a-3p detection compared to the non-specific fluorescent dye used in Fig. 2b .", "2b . As the general trend was suggestive of a synergistic induction of miR-200a-3p in response to mixed infection (Fig. 3a) , we hypothesized microRNA-200a-3p may play a role in the regulation of CXCL10 (IP-10), which was also synergistically upregulated in mixed-infected MDMs ( Fig.", "3a) , we hypothesized microRNA-200a-3p may play a role in the regulation of CXCL10 (IP-10), which was also synergistically upregulated in mixed-infected MDMs ( Fig. 2c and d) and primary HAEC ( Statistical analyses were performed using two-way ANOVA with Tukey's post-hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 CXCL10 (Fig.", "Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 CXCL10 (Fig. 3d) . These results suggested miR-200a-3p indirectly regulates CXCL10 and led us to hypothesize that miR-200a-3p controls a potential repressor of the JAK-STAT signaling pathway. . At 18 h after transfection, the MDMs were singly or mixed infected as described previously.", "At 18 h after transfection, the MDMs were singly or mixed infected as described previously. At 8 h post-IAV and/or SP infection, total mRNA was extracted and amplified by PCR using specific primers for the indicated genes. Values represent median ± IQR (a, c) or mean ± SEM (d, e) of three biological replicates.", "Values represent median ± IQR (a, c) or mean ± SEM (d, e) of three biological replicates. Statistical analyses were performed using a Kruskal-Wallis test (non-parametric, one-way ANOVA with Dunn's post-hoc test) for data presented in (a, c). An ordinary two-way ANOVA (with Tukey's post-hoc multiple comparison test) was used for data presented in (d, e).", "An ordinary two-way ANOVA (with Tukey's post-hoc multiple comparison test) was used for data presented in (d, e). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. MiRNA-200a-3p indirectly regulates IP-10 expression by targeting SOCS6.", "MiRNA-200a-3p indirectly regulates IP-10 expression by targeting SOCS6. As shown in Fig. 2a , several JAK-STAT signaling pathway genes were deregulated in mixed IAV-and SP-infected human MDMs; therefore, we hypothesized that miR-200a-3p directly regulates a regulator of the JAK-STAT signaling pathway.", "2a , several JAK-STAT signaling pathway genes were deregulated in mixed IAV-and SP-infected human MDMs; therefore, we hypothesized that miR-200a-3p directly regulates a regulator of the JAK-STAT signaling pathway. Predictive target analysis indicated that the 3' UTR of suppressor of cytokine signaling-6 (SOCS6) may be targeted by miR-200a-3p (Fig. 3b) .", "3b) . SOCS proteins constitute a class of negative regulators of JAK-STAT signaling pathways that are induced by both cytokines and TLR signaling. MiRNA-200a-3p was not predicted to target any of the other six members of the SOCS gene family.", "MiRNA-200a-3p was not predicted to target any of the other six members of the SOCS gene family. Transfection of human MDMs with MIM-200a downregulated SOCS6 (FC = 0.57) while inhibition of miR-200a-3p (INH-200a) upregulated SOCS6 (FC = 1.55), confirming that miR-200a-3p effectively regulates the expression of SOCS6 (Fig. 3e) .", "3e) . Moreover, SOCS6 was synergistically downregulated in IAV-or IAV/SP-infected MDMs overexpressing miRNA-200a (Fig. 3e) , suggesting that both infection and miR-200a-3p negatively regulate the expression of SOCS6. Finally, western blotting confirmed that expression of SOCS-6 sharply reduced following infection, especially after mixed IAV and SP infection (Fig. 3f) .", "3f) . These results indicate miR-200a-3p is strongly induced in response to mixed viral and bacterial co-infection, which in turn leads to downregulation of the JAK-STAT regulator SOCS-6 at both the mRNA and protein levels and subsequent upregulation of IP-10. analyses demonstrated mixed IAV and SP infection of human MDMs and HAEC induced significant production of IP-10.", "analyses demonstrated mixed IAV and SP infection of human MDMs and HAEC induced significant production of IP-10. As blood leukocytes and respiratory tract epithelial cells actively contribute to inflammation during pneumonia, we hypothesized the level of IP-10 in serum of patient with pneumonia may be both indicative of mixed respiratory infection and disease severity. As part of a prospective, hospital-based, multicenter case-control study on the etiology of pneumonia among children under 5-years-old, a total of 74 patients (44 male, 30 female) were included in this pilot evaluation.", "As part of a prospective, hospital-based, multicenter case-control study on the etiology of pneumonia among children under 5-years-old, a total of 74 patients (44 male, 30 female) were included in this pilot evaluation. According to WHO guidelines, retrospective analysis indicated 44 (59.5%) children had clinical signs of non-severe pneumonia and 30 (40.5%) children had signs of severe pneumonia. The main patient characteristics at inclusion are shown in Table 1 .", "The main patient characteristics at inclusion are shown in Table 1 . Patients with severe pneumonia had significant more recorded episodes of dyspnea (P < 0.001), cyanosis (P = 0.03), lower chest indrawing (P < 0.001), dullness to percussion (P < 0.001) and lethargy (P < 0.001) during chest examination than patient with non-severe pneumonia. Moreover, pleural effusions were significantly more observed among critically ill patients and the duration of hospitalization was significantly longer for the children with severe pneumonia than for those with non-severe pneumonia (P = 0.0015).", "Moreover, pleural effusions were significantly more observed among critically ill patients and the duration of hospitalization was significantly longer for the children with severe pneumonia than for those with non-severe pneumonia (P = 0.0015). Two deaths occurred within the group of children retrospectively defined with severe pneumonia. Evaluation of the systemic inflammatory response of the 74 cases is shown in Table 2 .", "Evaluation of the systemic inflammatory response of the 74 cases is shown in Table 2 . Serum level of CRP, IP-10, PCT, G-CSF, IL-6, IL-8 and MIP-1β were significantly more elevated in serum samples from critically ill patients. Patients with severe pneumonia had significantly higher (4.2-fold) serum IP-10 levels than those with a non-severe pneumonia (P < 0.001) suggesting IP-10 as a promising prognostic marker in pneumonia.", "Patients with severe pneumonia had significantly higher (4.2-fold) serum IP-10 levels than those with a non-severe pneumonia (P < 0.001) suggesting IP-10 as a promising prognostic marker in pneumonia. Diagnostic accuracy measures for predicting pneumonia severity using blood-based biomarkers are summarized in Table S3 . Briefly, in this study, the optimal IP-10 cut-off value for identifying patient with severe pneumonia was 4,240 pg ml −1 , with an area under the receiver operating characteristic curve of 0.69 (95% CI, 0.57 to 0.82, P < 0.001).", "Briefly, in this study, the optimal IP-10 cut-off value for identifying patient with severe pneumonia was 4,240 pg ml −1 , with an area under the receiver operating characteristic curve of 0.69 (95% CI, 0.57 to 0.82, P < 0.001). Defining as positive a serum IP-10 level above this cut-off resulted in a sensitivity of 63.3%, specificity of 63.6% and a positive likelihood ratio of 1.74. Prognostic values of IP-10 were closed to procalcitonin (PCT; AUC = 0.70; 95% IC, 0.58 to 0.82, P < 0.001) and IL-6 (AUC = 0.70; 95% IC, 0.58-0.83, P < 0.001).", "Prognostic values of IP-10 were closed to procalcitonin (PCT; AUC = 0.70; 95% IC, 0.58 to 0.82, P < 0.001) and IL-6 (AUC = 0.70; 95% IC, 0.58-0.83, P < 0.001). Multiplex PCR-based screening of respiratory and blood samples reveal a high variety of pathogen associations (Table 3) . Respiratory viruses were detected in the nasal aspirates (NAs) of 63/74 patients (85.1%).", "Respiratory viruses were detected in the nasal aspirates (NAs) of 63/74 patients (85.1%). Etiological bacteria of pneumonia (S. pneumoniae, n = 19; S. aureus, n = 1; or H. influenzae type B, n = 7) were identified via real-time PCR in the blood samples of 27/74 (36.5%) of the patients. Multiplex PCR assays allowed the identification of respiratory bacteria in the blood of 19 patients with negative blood culture results.", "Multiplex PCR assays allowed the identification of respiratory bacteria in the blood of 19 patients with negative blood culture results. Among the 74 cases PCR-positive for respiratory pathogens, a single virus or bacteria were detected in the NAs of 7 (9.4%) and 3 (4.0%) patients, respectively; these 10/74 (13.5%) cases were defined as the single infection group. The mixed infection group included the 62/74 (83.8%) cases in which (1) multiple viruses and/or bacteria were identified in NAs (38/74; 51.3%) without any bacteria identified in blood samples or (2) one or more viruses and/or bacteria were identified in NAs and associated with a blood bacteremia (24/74; 32.4%).", "The mixed infection group included the 62/74 (83.8%) cases in which (1) multiple viruses and/or bacteria were identified in NAs (38/74; 51.3%) without any bacteria identified in blood samples or (2) one or more viruses and/or bacteria were identified in NAs and associated with a blood bacteremia (24/74; 32.4%). We evaluated whether IP-10 serum level could correlate with the viral and bacterial etiologies of pneumonia. Patients with mixed infection had significant higher (3.6-fold) IP-10 serum level than patient with single detection (P = 0.03; Table 4 ).", "Patients with mixed infection had significant higher (3.6-fold) IP-10 serum level than patient with single detection (P = 0.03; Table 4 ). A stratified analysis reveals that the highest IP-10 serum level was observed among patients with both several respiratory pathogens identified (mixed-detection group) and severe pneumonia (14,427 pg ml −1 , IQR (3,981-82,994). In detail, a remarkable IP-10 serum level (142,531 pg ml −1 ), representing 33-fold higher above cut-off value predicting pneumonia severity was observed in patient with hRV in NA co-detected with S. pneumoniae (serotype 14) in pleural effusion and blood.", "In detail, a remarkable IP-10 serum level (142,531 pg ml −1 ), representing 33-fold higher above cut-off value predicting pneumonia severity was observed in patient with hRV in NA co-detected with S. pneumoniae (serotype 14) in pleural effusion and blood. In concordance with our in-vitro model of co-infection, a significant IP-10 level (90,338 pg ml −1 ) was quantified in blood sample of patient with severe bacteremic pneumococcal (serotype 14) pneumonia with a positive co-detection of Influenza B virus in NA. Taken together, these results suggest that high serum IP-10 levels are significantly associated with mixed viral and bacterial detection and also related to pneumonia pathogenesis.", "Taken together, these results suggest that high serum IP-10 levels are significantly associated with mixed viral and bacterial detection and also related to pneumonia pathogenesis. This study provides additional in vitro and clinical data to improve our understanding of the immunopathology of mixed viral and bacterial pneumonia (Fig. 4) .", "4) . The in vitro model of influenza and pneumococcal superinfection of human MDMs demonstrated that mixed infection synergistically induced release of the pro-inflammatory chemokine IP-10, strongly suggesting human Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 blood leukocytes contribute to the immunopathology of pneumonia. Additionally, transcriptomics and omics analyses provided new data on the inflammatory pathways that are activated during mixed infection and related to synergistic induction of the pro-inflammatory chemokine IP-10 in mixed infected cells.", "Additionally, transcriptomics and omics analyses provided new data on the inflammatory pathways that are activated during mixed infection and related to synergistic induction of the pro-inflammatory chemokine IP-10 in mixed infected cells. Our observations are consistent with a recent study describing IP-10 induction as host-proteome signature of both viral and bacterial infections 30 . Of the differentially-expressed genes observed in mixed infected MDMs, the transcription factors STAT-1 and IRF-7 appear to play crucial roles in the regulation of interferon-stimulated genes including CXCL10 (IP-10).", "Of the differentially-expressed genes observed in mixed infected MDMs, the transcription factors STAT-1 and IRF-7 appear to play crucial roles in the regulation of interferon-stimulated genes including CXCL10 (IP-10). By focusing on the intracellular mechanisms that regulate inflammatory pathways, we demonstrated a novel role for miRNA-200a-3p in the regulation of CXCL10 (IP-10). These observations are consistent with previous reports showing that RNA virus infection upregulates miR-155 in macrophages and dendritic cells and also regulates suppressor of cytokine signaling 1 (SOCS1), suggesting the existence of a miRNA/JAK-STAT/SOCS regulatory pathway during viral infection 29 .", "These observations are consistent with previous reports showing that RNA virus infection upregulates miR-155 in macrophages and dendritic cells and also regulates suppressor of cytokine signaling 1 (SOCS1), suggesting the existence of a miRNA/JAK-STAT/SOCS regulatory pathway during viral infection 29 . Our study suggests co-infection leads to overexpression of miR-200a-3p, which in turn targets and downregulates the JAK-STAT regulator SOCS-6 and consequently increases CXCL10 (IP-10) expression. Interestingly, a complementary in-silico approach reveals that several microRNAs that were found dysregulated in our experiments of IAV and SP co-infection of MDMs or HAEC, might target several genes of SOCS family and play similar role than miR-200a-3p.", "Interestingly, a complementary in-silico approach reveals that several microRNAs that were found dysregulated in our experiments of IAV and SP co-infection of MDMs or HAEC, might target several genes of SOCS family and play similar role than miR-200a-3p. Indeed, miRNA-142-3p might target SOCS4, 5, 6 mRNA while miRNA-194-5p might target SOCS2, 3, 4, 5 and 7 mRNA. These observations underline that intra-cellular regulation of IP-10 is not limited to the contribution of a sole microRNA.", "These observations underline that intra-cellular regulation of IP-10 is not limited to the contribution of a sole microRNA. A complex inter-relationship between numerous host microRNAs and inhibitors of the JAK-STAT signaling pathway occur to control host innate inflammatory response against viral and/or bacterial infections. Clinically, the majority of pediatric CAP cases in this study were associated with both positive viral and/or bacterial detection.", "Clinically, the majority of pediatric CAP cases in this study were associated with both positive viral and/or bacterial detection. Respiratory microorganisms were detected in 97% of cases; 51.3% of which were viral-viral, viral-bacterial or bacterial-bacterial co-detected only in nasal aspirates, 32.4% of which co-detected in both nasal aspirates and blood samples. These data are consistent with previous etiological studies of pediatric CAP 3,31-33 .", "These data are consistent with previous etiological studies of pediatric CAP 3,31-33 . S. pneumoniae was the major bacteria identified in blood (19/74; 25.7%) and mainly co-detected with respiratory viruses in NAs (16/19; 84.2%). We observed a very high diversity of viral and bacterial associations in biological samples from children with pneumonia.", "We observed a very high diversity of viral and bacterial associations in biological samples from children with pneumonia. In comparison with IAV and SP14 combination evaluated in-vitro, no pneumonia cases were singly influenza and pneumococcus infected, and no similar co-detection with those two pathogens has been clinically observed. Nevertheless, Influenza B (IVB) virus was identified in 5 patients and two of them had a positive SP co-detection in blood (one non-typable strain and one serotype 14 using our molecular typing test).", "Nevertheless, Influenza B (IVB) virus was identified in 5 patients and two of them had a positive SP co-detection in blood (one non-typable strain and one serotype 14 using our molecular typing test). IVB and SP14 combination seems to be the nearest pathogen co-detection to that in-vitro investigated. Clinically, this co-detection was associated with both a very high IP-10 expression and a very severe pneumonia case definition.", "Clinically, this co-detection was associated with both a very high IP-10 expression and a very severe pneumonia case definition. Interestingly, our translational pilot evaluation reveals IP-10 expression can be induced by several different viral and/or bacterial combinations. As immune response to each pathogen is different, further in-vitro investigations using different pathogens associations are needed to better characterize the mechanisms involved in the immunopathology of pneumonia.", "As immune response to each pathogen is different, further in-vitro investigations using different pathogens associations are needed to better characterize the mechanisms involved in the immunopathology of pneumonia. In this cohort, highest serum IP-10 levels were identified among patients with both several pathogen detected and severe pneumonia, suggesting a significant role of IP-10 on pneumonia pathogenesis. Indeed, high plasma levels of IP-10 have previously been reported in patients with sepsis 12 , and were associated with high mortality rate, especially among patients with CAP 34 .", "Indeed, high plasma levels of IP-10 have previously been reported in patients with sepsis 12 , and were associated with high mortality rate, especially among patients with CAP 34 . Additionally, the IP-10-CXCR3 axis has been related to acute immune lung injury and lymphocyte apoptosis during the development of severe acute respiratory syndrome (SARS) 35, 36 . Moreover, an in vivo study that modeled influenza and pneumococcal superinfection in mice indicated that pro-inflammatory chemokines, including IP-10, play a crucial role in influenza-induced susceptibility to lung neutrophilia, severe immunopathology and mortality 37 .", "Moreover, an in vivo study that modeled influenza and pneumococcal superinfection in mice indicated that pro-inflammatory chemokines, including IP-10, play a crucial role in influenza-induced susceptibility to lung neutrophilia, severe immunopathology and mortality 37 . In this study, markedly elevated IP-10 (92,809 pg ml −1 ) combined with the highest PCT level (74.4 pg ml −1 ) were quantified in the serum sample of a child who died, in whom S. pneumoniae (serotype 9 V) was identified in the blood (PCR and blood culture) and co-detected with Haemophilus influenzae type B in nasal aspirate. These observations suggest an interrelationship between co-detection, elevated serum IP-10 and the pathogenesis of pneumonia.", "These observations suggest an interrelationship between co-detection, elevated serum IP-10 and the pathogenesis of pneumonia. Several limitations of this pilot translational study need to be acknowledged before concluding mixed infection is related to elevated IP-10 and disease severity. Indeed, although viral shedding (e.g., of HRV and HBoV) is common in asymptomatic children, we were unable to evaluate the levels of immunomodulators in the serum samples of a control group.", "Indeed, although viral shedding (e.g., of HRV and HBoV) is common in asymptomatic children, we were unable to evaluate the levels of immunomodulators in the serum samples of a control group. Moreover, although the samples were collected within the first 24 hours after admission, only a single blood sample was processed for each patient. Therefore, a larger, longitudinal study on the etiology and severity of pneumonia will be necessary to confirm these results.", "Therefore, a larger, longitudinal study on the etiology and severity of pneumonia will be necessary to confirm these results. In conclusion, the present findings suggest that mixed respiratory infections and IP-10 may play major, interconnected roles in the pathogenesis of pneumonia. Clinically, assessment and monitoring of induced IP-10 serum level may assist clinicians to improve diagnosis and patient management of severe community-acquired pneumonia.", "Clinically, assessment and monitoring of induced IP-10 serum level may assist clinicians to improve diagnosis and patient management of severe community-acquired pneumonia. Viral and bacterial strains. The 10 ng ml −1 M-CSF (Miltenyi Biotec).", "Viral and bacterial strains. The 10 ng ml −1 M-CSF (Miltenyi Biotec). THP− 1 MDMs were obtained by culturing cells with 10 ng ml -1 phorbol myristate acetate (PMA; Invivogen, Toulouse, France) for 72 hours.", "THP− 1 MDMs were obtained by culturing cells with 10 ng ml -1 phorbol myristate acetate (PMA; Invivogen, Toulouse, France) for 72 hours. Human airway epithelial cells (HAEC, bronchial cell type) originated from a 54-years old woman with no pathology reported (batch number MD056501) were provided by Mucilair (Epithelix, Geneva, Switzerland). Sterility, tissue integrity (TEER), mucus production and cilia beating frequency have been certified by the company.", "Sterility, tissue integrity (TEER), mucus production and cilia beating frequency have been certified by the company. Gene expression profiling. Total cellular mRNA was purified using the RNeasy kit (Qiagen, Hilden, Germany). Reverse-transcription of total mRNA was performed using the RT 2 First Strand Kit (SABiosciences, Hilden, Germany).", "Reverse-transcription of total mRNA was performed using the RT 2 First Strand Kit (SABiosciences, Hilden, Germany). The expression of 84 genes involved in the human innate and adaptive immune responses was evaluated using the RT 2 profiler ™ PCR Array (SABiosciences) according to the manufacturer's recommendations. The Δ Δ Ct method was applied to calculate the fold changes in gene expression for each gene relative to uninfected control cells using the web-based RT 2 profiler PCR Array Data Analysis software (SABiosciences).", "The Δ Δ Ct method was applied to calculate the fold changes in gene expression for each gene relative to uninfected control cells using the web-based RT 2 profiler PCR Array Data Analysis software (SABiosciences). MicroRNA profiling array. Total cellular microRNAs were purified using the miRNeasy Mini kit (Qiagen) and reverse-transcribed using the miScript Reverse Transcription kit (Qiagen).", "Total cellular microRNAs were purified using the miRNeasy Mini kit (Qiagen) and reverse-transcribed using the miScript Reverse Transcription kit (Qiagen). The profiling of 84 miRNAs was performed using the Human Immunopathology miScript miRNA PCR Array kit (Qiagen) according to the manufacturer's instructions. Data were analyzed using the miScript miRNA PCR array data analysis web portal.", "Data were analyzed using the miScript miRNA PCR array data analysis web portal. In silico miRNA target prediction. MiRNA target genes were retrieved and compiled using TargetScan 38 and microRNA.org resource 39 . The interactions between miRNAs and intracellular pathways were predicted using DIANA-miRPath v2.0 40 .", "The interactions between miRNAs and intracellular pathways were predicted using DIANA-miRPath v2.0 40 . THP-1 MDMs were seeded in 24-well plates (0.5 × 10 6 per well) in triplicate, exposed to Influenza A H1N1 (A/Solomon islands/3/2006) virus (IAV) under serum-free conditions for 1 hour and then cultured for 3 hours in fresh RPMI-1640 containing 2% FBS. Streptococcus pneumoniae (SP) serotype 14 was added at 4 hours after IAV infection.", "Streptococcus pneumoniae (SP) serotype 14 was added at 4 hours after IAV infection. Gentamicin (10 μ g ml −1 ) was added 2 hours after SP infection (i.e. 6 hours post-influenza infection) and maintained in the culture media throughout the experiment to kill extracellular bacteria and limit bacterial growth.", "6 hours post-influenza infection) and maintained in the culture media throughout the experiment to kill extracellular bacteria and limit bacterial growth. Cell viability was determined by flow-cytometry using the FITC/Annexin V apoptosis detection kit (BD Biosciences), according to the manufacturer's instructions. #4427975) .", "#4427975) . In this assay, fold changes have been defined by the Δ Δ Ct method using control RNU-44 and -48 as reference microRNAs. Total mRNA was purified from transfected and infected MDMs using the RNeasy kit (Qiagen) and specific primers were used to amplify transforming growth factor beta-2 (TGFB2; F: 5′ -CCATCCCGCCCACTTTCTAC-3′ , R: 5′ -AGCTCAATCCGTTGTTCAGGC-3′ ), SOCS6 (F: 5′ -AAGAATTCATCCCTTGGATTAGGTAAC-3′ , R: 5′ -CAGACTGGAGGTCGTGGAA-3′ ) 41 43 , and 3) absence of wheezing at auscultation, and, 4) first symptoms appearing within the last 14 days, and 5) radiological confirmation of pneumonia as per WHO guidelines 44 .", "Total mRNA was purified from transfected and infected MDMs using the RNeasy kit (Qiagen) and specific primers were used to amplify transforming growth factor beta-2 (TGFB2; F: 5′ -CCATCCCGCCCACTTTCTAC-3′ , R: 5′ -AGCTCAATCCGTTGTTCAGGC-3′ ), SOCS6 (F: 5′ -AAGAATTCATCCCTTGGATTAGGTAAC-3′ , R: 5′ -CAGACTGGAGGTCGTGGAA-3′ ) 41 43 , and 3) absence of wheezing at auscultation, and, 4) first symptoms appearing within the last 14 days, and 5) radiological confirmation of pneumonia as per WHO guidelines 44 . Based on these primary criteria defining pneumonia cases, all 74 cases were retrospectively re-evaluated according to the WHO \"Pocket book of hospital care for children\" 45 criteria to evaluate pneumonia severity. Cases that died during the study, or who had at least one additional clinical signs including central cyanosis, dullness to percussion during chest examination, prostration/lethargy, pleural effusion observed on chest radiography were retrospectively included in the severe pneumonia group.", "Cases that died during the study, or who had at least one additional clinical signs including central cyanosis, dullness to percussion during chest examination, prostration/lethargy, pleural effusion observed on chest radiography were retrospectively included in the severe pneumonia group. Patients without any of these additional clinical signs were included in the non-severe pneumonia group. Table 4 .", "Table 4 . a IP-10 values are expressed in pg ml -1 . IP-10 concentration differences between groups were compared using unpaired Mann-Whitney tests; significant changes (P < 0.05) are in bold. Clinical and molecular analysis. Nasopharyngeal aspirates (NAs) and whole blood samples were collected from children within 24 hours of admission.", "Nasopharyngeal aspirates (NAs) and whole blood samples were collected from children within 24 hours of admission. Whole blood samples were used for complete blood counts, blood culture and multiplex real-time PCR to identify Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae type B 46 . S. pneumoniae serotypes were defined using a 11 multiplex real-time PCR assay targeting the 40 most frequently represented serotypes or serogroups according to protocol developed by Messaoudi et al.", "S. pneumoniae serotypes were defined using a 11 multiplex real-time PCR assay targeting the 40 most frequently represented serotypes or serogroups according to protocol developed by Messaoudi et al. 47 . Serum C-reactive protein (CRP; AssayPro, St. Charles, Missouri, United States) and Procalcitonin (PCT; VIDAS B.R.A.H.M.S; bioMérieux) were quantified from whole-blood samples.", "Serum C-reactive protein (CRP; AssayPro, St. Charles, Missouri, United States) and Procalcitonin (PCT; VIDAS B.R.A.H.M.S; bioMérieux) were quantified from whole-blood samples. Multiplex real-time non quantitative PCR (Fast-Track Diagnostic, Sliema, Malta) was used to detect 19 viruses and five bacteria in the respiratory specimens (NAs and pleural effusions). Mixed detection was defined as 1) PCR-positive for multiple viruses in NAs, 2) positive blood culture or PCR-positive for multiple bacteria in blood or 3) PCR-positive for one or multiple viruses in NAs and one or multiple bacteria in blood (identified by PCR and blood culture).", "Mixed detection was defined as 1) PCR-positive for multiple viruses in NAs, 2) positive blood culture or PCR-positive for multiple bacteria in blood or 3) PCR-positive for one or multiple viruses in NAs and one or multiple bacteria in blood (identified by PCR and blood culture). Ethical approval. The study protocol, informed consent statement, clinical research form, any amendments and all other study documents were submitted to and approved by the Ethical Committee of the Instituto de Investigaciones en Ciencias de la Salud, the Universidad Nacional de Asunción (IICS-UNA) and the Hospital Pediátrico Niños de Acosta Ñu.", "The study protocol, informed consent statement, clinical research form, any amendments and all other study documents were submitted to and approved by the Ethical Committee of the Instituto de Investigaciones en Ciencias de la Salud, the Universidad Nacional de Asunción (IICS-UNA) and the Hospital Pediátrico Niños de Acosta Ñu. Informed consent was obtained from all subjects involved in this study. The clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki.", "The clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki. Statistical analysis. The Chi-square test and Fisher's exact test were used to compare categorical variables; continuous variables and non-normally distributed data were compared using the Mann-Whitney U-test; normally distributed data were compared using unpaired t-tests.", "The Chi-square test and Fisher's exact test were used to compare categorical variables; continuous variables and non-normally distributed data were compared using the Mann-Whitney U-test; normally distributed data were compared using unpaired t-tests. Comparative analyses between experimental conditions (i.e., MOCK, IAV, SP or IAV + SP) were performed using one-way ANOVA with Tukey's post-hoc test or Kruskal-Wallis analysis with Dunn's post-hoc tests. Receiver operating curve (ROC) analysis was used to determine the optimal cut-off value for IP-10 to differentiate between non-severe and severe pneumonia cases.", "Receiver operating curve (ROC) analysis was used to determine the optimal cut-off value for IP-10 to differentiate between non-severe and severe pneumonia cases. P < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism (La Jolla, California, United States)." ]

[ "Mixed viral and bacterial infections are widely described in community-acquired pneumonia; however, the clinical implications of co-infection on the associated immunopathology remain poorly studied. In this study, microRNA, mRNA and cytokine/chemokine secretion profiling were investigated for human monocyte-derived macrophages infected in-vitro with Influenza virus A/H1N1 and/or Streptococcus pneumoniae. We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions.", "We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions. We demonstrated that endogenous miRNA-200a-3p, whose expression was synergistically induced following co-infection, indirectly regulates CXCL10 expression by targeting suppressor of cytokine signaling-6 (SOCS-6), a well-known regulator of the JAK-STAT signaling pathway. Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5 years-old) hospitalized with viral and/or bacterial community-acquired pneumonia.", "Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5 years-old) hospitalized with viral and/or bacterial community-acquired pneumonia. Clinically, among the 74 cases of pneumonia, patients with identified mixed-detection had significantly higher (3.6-fold) serum IP-10 levels than those with a single detection (P = 0.03), and were significantly associated with severe pneumonia (P < 0.01). This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia.", "This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia. Text: Scientific RepoRts | 6:38532 | DOI: 10 .1038/srep38532 pathogenesis of several diseases and has been suggested as a potential biomarker of viral infection 10, 11 , late-onset bacterial infection in premature infants 12 , and a promising biomarker of sepsis and septic shock 13, 14 . Combined analysis of IP-10 and IFN-γ has also been reported as a useful biomarker for diagnosis and monitoring therapeutic efficacy in patients with active tuberculosis [15] [16] [17] , and both remain detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunction 18 .", "Combined analysis of IP-10 and IFN-γ has also been reported as a useful biomarker for diagnosis and monitoring therapeutic efficacy in patients with active tuberculosis [15] [16] [17] , and both remain detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunction 18 . With airway epithelial cells 19 , resident alveolar macrophages (AMs) and blood monocytes-derived macrophages (recruited into tissues under inflammatory conditions 20, 21 ) represent a major line of defense against both pneumococcal (through their high phagocytic capacity [22] [23] [24] ) and influenza infection 25, 26 . So far, no studies have yet focused on the intracellular mechanisms that regulate IP-10 in human blood leukocytes during mixed IAV and SP infection.", "So far, no studies have yet focused on the intracellular mechanisms that regulate IP-10 in human blood leukocytes during mixed IAV and SP infection. Several studies indicated that host non-coding small RNAs (including microRNAs) may function as immunomodulators by regulating several pivotal intracellular processes, such as the innate immune response 27 and antiviral activity 28, 29 ; both of these processes are closely related to toll-like receptor (TLR) signaling pathways. In this study, we firstly investigated the in vitro intracellular mechanisms that mediate the innate immune response in IAV and/or SP infected human monocyte-derived macrophages (MDMs).", "In this study, we firstly investigated the in vitro intracellular mechanisms that mediate the innate immune response in IAV and/or SP infected human monocyte-derived macrophages (MDMs). Using this approach, we observed that mixed-infection of MDMs induces a synergistic production of IP-10 which can be related to a miRNA-200a/JAK-STAT/SOCS-6 regulatory pathway. Subsequently, in a retrospective analysis of clinical samples collected from children ≤ 5 years-old hospitalized with pneumonia, we confirmed that serum IP-10 level could be related to both viral and/or bacterial etiologies and disease severity.", "Subsequently, in a retrospective analysis of clinical samples collected from children ≤ 5 years-old hospitalized with pneumonia, we confirmed that serum IP-10 level could be related to both viral and/or bacterial etiologies and disease severity. Characteristics of MDMs infected by IAV and/or SP. Initially, we investigated in vitro the impact of single and mixed IAV and SP infection on MDMs.", "Initially, we investigated in vitro the impact of single and mixed IAV and SP infection on MDMs. Firstly, active replication of IAV was assessed by qRT-PCR and quantification of new infectious viral particles in the cell supernatants ( Fig. 1a,b ).", "1a,b ). IAV titer increased over time after single infection with IAV and correlated with increased production of negative-strand IAV RNA. Maximum viral replication was observed at 18-24 hours post-infection, after which time both RNA replication and the quantity of infectious particles decreased.", "Maximum viral replication was observed at 18-24 hours post-infection, after which time both RNA replication and the quantity of infectious particles decreased. In this in vitro model, subsequent challenge of IAV-infected MDMs with SP had no significant impact on the production of new infectious viral particles (Fig. 1b) .", "1b) . Together, these results indicate permissive and productive infection of MDMs by IAV. Secondly, we evaluated whether MDMs are permissive for both IAV and SP infection. The presence of pneumococci within IAV-and SP-infected primary MDMs was confirmed at 8 h post-infection (Fig.", "The presence of pneumococci within IAV-and SP-infected primary MDMs was confirmed at 8 h post-infection (Fig. 1c) , suggesting that MDMs are permissive for viral and bacterial co-infection in the early steps of infection. Importantly, confocal co-detection of mixed IAV and SP was only effective following 8 h post-infection due to the bactericidal impact of SP internalization within human macrophages (after 24 h, data not shown).", "Importantly, confocal co-detection of mixed IAV and SP was only effective following 8 h post-infection due to the bactericidal impact of SP internalization within human macrophages (after 24 h, data not shown). Thirdly, we evaluated the impact of single and mixed infection with IAV and SP on MDM viability. Mixed infection significantly decreased cell viability (65.2 ± 4.5% total cell death at 48 hours post-infection; P < 0.0001) compared to single SP and IAV infection (39.6 ± 1.7% and 17.4 ± 1.1% total cell death, respectively; Fig.", "Mixed infection significantly decreased cell viability (65.2 ± 4.5% total cell death at 48 hours post-infection; P < 0.0001) compared to single SP and IAV infection (39.6 ± 1.7% and 17.4 ± 1.1% total cell death, respectively; Fig. 1d ). Taken together, these results confirmed human MDMs are permissive to mixed viral and bacterial infection.", "Taken together, these results confirmed human MDMs are permissive to mixed viral and bacterial infection. mRNA, microRNA and protein expression profiling reveal an overall induction of the host innate immune response following IAV and/or SP infection of MDMs. To investigate the innate immune response orchestrated by IAV-and SP-infected human MDMs, we firstly evaluated the expression of 84 genes involved in the innate and adaptive immune responses (Table S1) ; the major differentially-expressed genes are summarized in Fig.", "To investigate the innate immune response orchestrated by IAV-and SP-infected human MDMs, we firstly evaluated the expression of 84 genes involved in the innate and adaptive immune responses (Table S1) ; the major differentially-expressed genes are summarized in Fig. 2a . Expression profiling indicated an overall induction of genes related to the JAK-STAT, NF-Κ β and TLR signaling pathways.", "Expression profiling indicated an overall induction of genes related to the JAK-STAT, NF-Κ β and TLR signaling pathways. Indeed, all interferon-stimulated genes (ISGs) screened, including CXCL10 (fold-change [FC] = 240.9), CCL-2 (FC = 34.2) and MX-1 (FC = 151.4) were upregulated following mixed infection compared to uninfected cells, most of which are closely related to STAT-1 (FC = 52.3), IRF-7 (FC = 6.8) and IFNB1 (FC = 5.2) also found upregulated in mixed infected cells. Secondly, we investigated the endogenous microRNA expression profiles of IAV-and SP-infected MDMs.", "Secondly, we investigated the endogenous microRNA expression profiles of IAV-and SP-infected MDMs. A selection of microRNAs that were found to be differentially-expressed under different infection conditions are shown in Fig. 2b and Table S2 .", "2b and Table S2 . MiRNA-200a-3p was overexpressed after both single IAV (FC = 6.9), single SP (FC = 3.7) and mixed IAV/SP infection (FC = 7.3), indicating this miRNA may play a role in the innate immune response to viral and bacterial co-infection. Similar miRNA-200a-3p dysregulation profiles were obtained following IAV and/or SP infections of human macrophages-like (THP-1 monocytes-derived macrophages) or primary MDMs (data not shown).", "Similar miRNA-200a-3p dysregulation profiles were obtained following IAV and/or SP infections of human macrophages-like (THP-1 monocytes-derived macrophages) or primary MDMs (data not shown). Thirdly, the secreted levels of various antiviral, pro-inflammatory and immunomodulatory cytokines/chemokines were assayed in IAV-and SP-infected-THP-1 and primary MDM cell supernatants. We observed a remarkable correlation between the mRNA and protein expression profiles of single or mixed infected MDMs especially regarding CXCL-10 and IP-10 expression.", "We observed a remarkable correlation between the mRNA and protein expression profiles of single or mixed infected MDMs especially regarding CXCL-10 and IP-10 expression. Indeed, the level of IP-10 was synergistically increased in the supernatant of IAV-infected THP-1 MDMs exposed to SP (mean: 30,589 ± 16,484 pg ml −1 ) compared to single IAV infection (1,439 ± 566.5 pg ml −1 ) and single SP infection (4,472 ± 2,001 pg ml −1 ; P≤ 0.05; Fig. 2c ) at 24 hours after infection.", "2c ) at 24 hours after infection. In those cells, IP-10 expression reduced over time (48 to 72 hours), coinciding with a significant higher proportion of necrotic and apoptotic cells (Fig. 1d) . The synergistic expression of IP-10 was similarly observed at 24 hours post-infection using primary MDMs (Fig. 2d) .", "2d) . Significantly increased secretion of the other tested cytokines and chemokines was not observed post-infection, even in mixed infected MDMs (Fig. S1 ). Interestingly, a significant production of IP-10 was also observed in supernatants of primary human airway epithelial cells (HAEC) mixed-infected by IAV and SP compared to the single infections (Fig.", "Interestingly, a significant production of IP-10 was also observed in supernatants of primary human airway epithelial cells (HAEC) mixed-infected by IAV and SP compared to the single infections (Fig. 2e) . Taken together, the mRNA and protein profiling results suggested that mixed viral and bacterial infection of MDMs induces a synergistic pro-inflammatory response related to the type-1 interferon and JAK-STAT signaling pathways, with IP-10 as signature of IAV/SP co-infection.", "Taken together, the mRNA and protein profiling results suggested that mixed viral and bacterial infection of MDMs induces a synergistic pro-inflammatory response related to the type-1 interferon and JAK-STAT signaling pathways, with IP-10 as signature of IAV/SP co-infection. Among all microRNAs screened, miR-200a-3p was the most Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 overexpressed in IAV/SP co-infection of human MDMs. In the remainder of this study, we decided to investigate the interconnection between miR-200a-3p expression and the innate immune response.", "In the remainder of this study, we decided to investigate the interconnection between miR-200a-3p expression and the innate immune response. Endogenous miRNA-200a-3p expression correlates with CXCL10 (IP-10) induction following mixed IAV and SP infection of human MDMs. Using a specific Taqman probe assay targeting miR-200a-3p, we confirmed a significant upregulation of miR-200a-3p following mixed IAV and SP infection of human MDMs (Fig.", "Using a specific Taqman probe assay targeting miR-200a-3p, we confirmed a significant upregulation of miR-200a-3p following mixed IAV and SP infection of human MDMs (Fig. 3a) . In this experiment, a more marked up-regulation of miR-200a-3p was observed following IAV+ SP compared to results obtained previously (Fig. 2b) .", "2b) . This discrepancy has been attributed to the use of two different approaches to quantify miR-200a-3p expression. The use of a target-specific stem-loop reverse transcription primer in Fig. 3a allows a better sensitivity of miR-200a-3p detection compared to the non-specific fluorescent dye used in Fig. 2b .", "2b . As the general trend was suggestive of a synergistic induction of miR-200a-3p in response to mixed infection (Fig. 3a) , we hypothesized microRNA-200a-3p may play a role in the regulation of CXCL10 (IP-10), which was also synergistically upregulated in mixed-infected MDMs ( Fig.", "3a) , we hypothesized microRNA-200a-3p may play a role in the regulation of CXCL10 (IP-10), which was also synergistically upregulated in mixed-infected MDMs ( Fig. 2c and d) and primary HAEC ( Statistical analyses were performed using two-way ANOVA with Tukey's post-hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 CXCL10 (Fig.", "Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 CXCL10 (Fig. 3d) . These results suggested miR-200a-3p indirectly regulates CXCL10 and led us to hypothesize that miR-200a-3p controls a potential repressor of the JAK-STAT signaling pathway. . At 18 h after transfection, the MDMs were singly or mixed infected as described previously.", "At 18 h after transfection, the MDMs were singly or mixed infected as described previously. At 8 h post-IAV and/or SP infection, total mRNA was extracted and amplified by PCR using specific primers for the indicated genes. Values represent median ± IQR (a, c) or mean ± SEM (d, e) of three biological replicates.", "Values represent median ± IQR (a, c) or mean ± SEM (d, e) of three biological replicates. Statistical analyses were performed using a Kruskal-Wallis test (non-parametric, one-way ANOVA with Dunn's post-hoc test) for data presented in (a, c). An ordinary two-way ANOVA (with Tukey's post-hoc multiple comparison test) was used for data presented in (d, e).", "An ordinary two-way ANOVA (with Tukey's post-hoc multiple comparison test) was used for data presented in (d, e). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. MiRNA-200a-3p indirectly regulates IP-10 expression by targeting SOCS6.", "MiRNA-200a-3p indirectly regulates IP-10 expression by targeting SOCS6. As shown in Fig. 2a , several JAK-STAT signaling pathway genes were deregulated in mixed IAV-and SP-infected human MDMs; therefore, we hypothesized that miR-200a-3p directly regulates a regulator of the JAK-STAT signaling pathway.", "2a , several JAK-STAT signaling pathway genes were deregulated in mixed IAV-and SP-infected human MDMs; therefore, we hypothesized that miR-200a-3p directly regulates a regulator of the JAK-STAT signaling pathway. Predictive target analysis indicated that the 3' UTR of suppressor of cytokine signaling-6 (SOCS6) may be targeted by miR-200a-3p (Fig. 3b) .", "3b) . SOCS proteins constitute a class of negative regulators of JAK-STAT signaling pathways that are induced by both cytokines and TLR signaling. MiRNA-200a-3p was not predicted to target any of the other six members of the SOCS gene family.", "MiRNA-200a-3p was not predicted to target any of the other six members of the SOCS gene family. Transfection of human MDMs with MIM-200a downregulated SOCS6 (FC = 0.57) while inhibition of miR-200a-3p (INH-200a) upregulated SOCS6 (FC = 1.55), confirming that miR-200a-3p effectively regulates the expression of SOCS6 (Fig. 3e) .", "3e) . Moreover, SOCS6 was synergistically downregulated in IAV-or IAV/SP-infected MDMs overexpressing miRNA-200a (Fig. 3e) , suggesting that both infection and miR-200a-3p negatively regulate the expression of SOCS6. Finally, western blotting confirmed that expression of SOCS-6 sharply reduced following infection, especially after mixed IAV and SP infection (Fig. 3f) .", "3f) . These results indicate miR-200a-3p is strongly induced in response to mixed viral and bacterial co-infection, which in turn leads to downregulation of the JAK-STAT regulator SOCS-6 at both the mRNA and protein levels and subsequent upregulation of IP-10. analyses demonstrated mixed IAV and SP infection of human MDMs and HAEC induced significant production of IP-10.", "analyses demonstrated mixed IAV and SP infection of human MDMs and HAEC induced significant production of IP-10. As blood leukocytes and respiratory tract epithelial cells actively contribute to inflammation during pneumonia, we hypothesized the level of IP-10 in serum of patient with pneumonia may be both indicative of mixed respiratory infection and disease severity. As part of a prospective, hospital-based, multicenter case-control study on the etiology of pneumonia among children under 5-years-old, a total of 74 patients (44 male, 30 female) were included in this pilot evaluation.", "As part of a prospective, hospital-based, multicenter case-control study on the etiology of pneumonia among children under 5-years-old, a total of 74 patients (44 male, 30 female) were included in this pilot evaluation. According to WHO guidelines, retrospective analysis indicated 44 (59.5%) children had clinical signs of non-severe pneumonia and 30 (40.5%) children had signs of severe pneumonia. The main patient characteristics at inclusion are shown in Table 1 .", "The main patient characteristics at inclusion are shown in Table 1 . Patients with severe pneumonia had significant more recorded episodes of dyspnea (P < 0.001), cyanosis (P = 0.03), lower chest indrawing (P < 0.001), dullness to percussion (P < 0.001) and lethargy (P < 0.001) during chest examination than patient with non-severe pneumonia. Moreover, pleural effusions were significantly more observed among critically ill patients and the duration of hospitalization was significantly longer for the children with severe pneumonia than for those with non-severe pneumonia (P = 0.0015).", "Moreover, pleural effusions were significantly more observed among critically ill patients and the duration of hospitalization was significantly longer for the children with severe pneumonia than for those with non-severe pneumonia (P = 0.0015). Two deaths occurred within the group of children retrospectively defined with severe pneumonia. Evaluation of the systemic inflammatory response of the 74 cases is shown in Table 2 .", "Evaluation of the systemic inflammatory response of the 74 cases is shown in Table 2 . Serum level of CRP, IP-10, PCT, G-CSF, IL-6, IL-8 and MIP-1β were significantly more elevated in serum samples from critically ill patients. Patients with severe pneumonia had significantly higher (4.2-fold) serum IP-10 levels than those with a non-severe pneumonia (P < 0.001) suggesting IP-10 as a promising prognostic marker in pneumonia.", "Patients with severe pneumonia had significantly higher (4.2-fold) serum IP-10 levels than those with a non-severe pneumonia (P < 0.001) suggesting IP-10 as a promising prognostic marker in pneumonia. Diagnostic accuracy measures for predicting pneumonia severity using blood-based biomarkers are summarized in Table S3 . Briefly, in this study, the optimal IP-10 cut-off value for identifying patient with severe pneumonia was 4,240 pg ml −1 , with an area under the receiver operating characteristic curve of 0.69 (95% CI, 0.57 to 0.82, P < 0.001).", "Briefly, in this study, the optimal IP-10 cut-off value for identifying patient with severe pneumonia was 4,240 pg ml −1 , with an area under the receiver operating characteristic curve of 0.69 (95% CI, 0.57 to 0.82, P < 0.001). Defining as positive a serum IP-10 level above this cut-off resulted in a sensitivity of 63.3%, specificity of 63.6% and a positive likelihood ratio of 1.74. Prognostic values of IP-10 were closed to procalcitonin (PCT; AUC = 0.70; 95% IC, 0.58 to 0.82, P < 0.001) and IL-6 (AUC = 0.70; 95% IC, 0.58-0.83, P < 0.001).", "Prognostic values of IP-10 were closed to procalcitonin (PCT; AUC = 0.70; 95% IC, 0.58 to 0.82, P < 0.001) and IL-6 (AUC = 0.70; 95% IC, 0.58-0.83, P < 0.001). Multiplex PCR-based screening of respiratory and blood samples reveal a high variety of pathogen associations (Table 3) . Respiratory viruses were detected in the nasal aspirates (NAs) of 63/74 patients (85.1%).", "Respiratory viruses were detected in the nasal aspirates (NAs) of 63/74 patients (85.1%). Etiological bacteria of pneumonia (S. pneumoniae, n = 19; S. aureus, n = 1; or H. influenzae type B, n = 7) were identified via real-time PCR in the blood samples of 27/74 (36.5%) of the patients. Multiplex PCR assays allowed the identification of respiratory bacteria in the blood of 19 patients with negative blood culture results.", "Multiplex PCR assays allowed the identification of respiratory bacteria in the blood of 19 patients with negative blood culture results. Among the 74 cases PCR-positive for respiratory pathogens, a single virus or bacteria were detected in the NAs of 7 (9.4%) and 3 (4.0%) patients, respectively; these 10/74 (13.5%) cases were defined as the single infection group. The mixed infection group included the 62/74 (83.8%) cases in which (1) multiple viruses and/or bacteria were identified in NAs (38/74; 51.3%) without any bacteria identified in blood samples or (2) one or more viruses and/or bacteria were identified in NAs and associated with a blood bacteremia (24/74; 32.4%).", "The mixed infection group included the 62/74 (83.8%) cases in which (1) multiple viruses and/or bacteria were identified in NAs (38/74; 51.3%) without any bacteria identified in blood samples or (2) one or more viruses and/or bacteria were identified in NAs and associated with a blood bacteremia (24/74; 32.4%). We evaluated whether IP-10 serum level could correlate with the viral and bacterial etiologies of pneumonia. Patients with mixed infection had significant higher (3.6-fold) IP-10 serum level than patient with single detection (P = 0.03; Table 4 ).", "Patients with mixed infection had significant higher (3.6-fold) IP-10 serum level than patient with single detection (P = 0.03; Table 4 ). A stratified analysis reveals that the highest IP-10 serum level was observed among patients with both several respiratory pathogens identified (mixed-detection group) and severe pneumonia (14,427 pg ml −1 , IQR (3,981-82,994). In detail, a remarkable IP-10 serum level (142,531 pg ml −1 ), representing 33-fold higher above cut-off value predicting pneumonia severity was observed in patient with hRV in NA co-detected with S. pneumoniae (serotype 14) in pleural effusion and blood.", "In detail, a remarkable IP-10 serum level (142,531 pg ml −1 ), representing 33-fold higher above cut-off value predicting pneumonia severity was observed in patient with hRV in NA co-detected with S. pneumoniae (serotype 14) in pleural effusion and blood. In concordance with our in-vitro model of co-infection, a significant IP-10 level (90,338 pg ml −1 ) was quantified in blood sample of patient with severe bacteremic pneumococcal (serotype 14) pneumonia with a positive co-detection of Influenza B virus in NA. Taken together, these results suggest that high serum IP-10 levels are significantly associated with mixed viral and bacterial detection and also related to pneumonia pathogenesis.", "Taken together, these results suggest that high serum IP-10 levels are significantly associated with mixed viral and bacterial detection and also related to pneumonia pathogenesis. This study provides additional in vitro and clinical data to improve our understanding of the immunopathology of mixed viral and bacterial pneumonia (Fig. 4) .", "4) . The in vitro model of influenza and pneumococcal superinfection of human MDMs demonstrated that mixed infection synergistically induced release of the pro-inflammatory chemokine IP-10, strongly suggesting human Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 blood leukocytes contribute to the immunopathology of pneumonia. Additionally, transcriptomics and omics analyses provided new data on the inflammatory pathways that are activated during mixed infection and related to synergistic induction of the pro-inflammatory chemokine IP-10 in mixed infected cells.", "Additionally, transcriptomics and omics analyses provided new data on the inflammatory pathways that are activated during mixed infection and related to synergistic induction of the pro-inflammatory chemokine IP-10 in mixed infected cells. Our observations are consistent with a recent study describing IP-10 induction as host-proteome signature of both viral and bacterial infections 30 . Of the differentially-expressed genes observed in mixed infected MDMs, the transcription factors STAT-1 and IRF-7 appear to play crucial roles in the regulation of interferon-stimulated genes including CXCL10 (IP-10).", "Of the differentially-expressed genes observed in mixed infected MDMs, the transcription factors STAT-1 and IRF-7 appear to play crucial roles in the regulation of interferon-stimulated genes including CXCL10 (IP-10). By focusing on the intracellular mechanisms that regulate inflammatory pathways, we demonstrated a novel role for miRNA-200a-3p in the regulation of CXCL10 (IP-10). These observations are consistent with previous reports showing that RNA virus infection upregulates miR-155 in macrophages and dendritic cells and also regulates suppressor of cytokine signaling 1 (SOCS1), suggesting the existence of a miRNA/JAK-STAT/SOCS regulatory pathway during viral infection 29 .", "These observations are consistent with previous reports showing that RNA virus infection upregulates miR-155 in macrophages and dendritic cells and also regulates suppressor of cytokine signaling 1 (SOCS1), suggesting the existence of a miRNA/JAK-STAT/SOCS regulatory pathway during viral infection 29 . Our study suggests co-infection leads to overexpression of miR-200a-3p, which in turn targets and downregulates the JAK-STAT regulator SOCS-6 and consequently increases CXCL10 (IP-10) expression. Interestingly, a complementary in-silico approach reveals that several microRNAs that were found dysregulated in our experiments of IAV and SP co-infection of MDMs or HAEC, might target several genes of SOCS family and play similar role than miR-200a-3p.", "Interestingly, a complementary in-silico approach reveals that several microRNAs that were found dysregulated in our experiments of IAV and SP co-infection of MDMs or HAEC, might target several genes of SOCS family and play similar role than miR-200a-3p. Indeed, miRNA-142-3p might target SOCS4, 5, 6 mRNA while miRNA-194-5p might target SOCS2, 3, 4, 5 and 7 mRNA. These observations underline that intra-cellular regulation of IP-10 is not limited to the contribution of a sole microRNA.", "These observations underline that intra-cellular regulation of IP-10 is not limited to the contribution of a sole microRNA. A complex inter-relationship between numerous host microRNAs and inhibitors of the JAK-STAT signaling pathway occur to control host innate inflammatory response against viral and/or bacterial infections. Clinically, the majority of pediatric CAP cases in this study were associated with both positive viral and/or bacterial detection.", "Clinically, the majority of pediatric CAP cases in this study were associated with both positive viral and/or bacterial detection. Respiratory microorganisms were detected in 97% of cases; 51.3% of which were viral-viral, viral-bacterial or bacterial-bacterial co-detected only in nasal aspirates, 32.4% of which co-detected in both nasal aspirates and blood samples. These data are consistent with previous etiological studies of pediatric CAP 3,31-33 .", "These data are consistent with previous etiological studies of pediatric CAP 3,31-33 . S. pneumoniae was the major bacteria identified in blood (19/74; 25.7%) and mainly co-detected with respiratory viruses in NAs (16/19; 84.2%). We observed a very high diversity of viral and bacterial associations in biological samples from children with pneumonia.", "We observed a very high diversity of viral and bacterial associations in biological samples from children with pneumonia. In comparison with IAV and SP14 combination evaluated in-vitro, no pneumonia cases were singly influenza and pneumococcus infected, and no similar co-detection with those two pathogens has been clinically observed. Nevertheless, Influenza B (IVB) virus was identified in 5 patients and two of them had a positive SP co-detection in blood (one non-typable strain and one serotype 14 using our molecular typing test).", "Nevertheless, Influenza B (IVB) virus was identified in 5 patients and two of them had a positive SP co-detection in blood (one non-typable strain and one serotype 14 using our molecular typing test). IVB and SP14 combination seems to be the nearest pathogen co-detection to that in-vitro investigated. Clinically, this co-detection was associated with both a very high IP-10 expression and a very severe pneumonia case definition.", "Clinically, this co-detection was associated with both a very high IP-10 expression and a very severe pneumonia case definition. Interestingly, our translational pilot evaluation reveals IP-10 expression can be induced by several different viral and/or bacterial combinations. As immune response to each pathogen is different, further in-vitro investigations using different pathogens associations are needed to better characterize the mechanisms involved in the immunopathology of pneumonia.", "As immune response to each pathogen is different, further in-vitro investigations using different pathogens associations are needed to better characterize the mechanisms involved in the immunopathology of pneumonia. In this cohort, highest serum IP-10 levels were identified among patients with both several pathogen detected and severe pneumonia, suggesting a significant role of IP-10 on pneumonia pathogenesis. Indeed, high plasma levels of IP-10 have previously been reported in patients with sepsis 12 , and were associated with high mortality rate, especially among patients with CAP 34 .", "Indeed, high plasma levels of IP-10 have previously been reported in patients with sepsis 12 , and were associated with high mortality rate, especially among patients with CAP 34 . Additionally, the IP-10-CXCR3 axis has been related to acute immune lung injury and lymphocyte apoptosis during the development of severe acute respiratory syndrome (SARS) 35, 36 . Moreover, an in vivo study that modeled influenza and pneumococcal superinfection in mice indicated that pro-inflammatory chemokines, including IP-10, play a crucial role in influenza-induced susceptibility to lung neutrophilia, severe immunopathology and mortality 37 .", "Moreover, an in vivo study that modeled influenza and pneumococcal superinfection in mice indicated that pro-inflammatory chemokines, including IP-10, play a crucial role in influenza-induced susceptibility to lung neutrophilia, severe immunopathology and mortality 37 . In this study, markedly elevated IP-10 (92,809 pg ml −1 ) combined with the highest PCT level (74.4 pg ml −1 ) were quantified in the serum sample of a child who died, in whom S. pneumoniae (serotype 9 V) was identified in the blood (PCR and blood culture) and co-detected with Haemophilus influenzae type B in nasal aspirate. These observations suggest an interrelationship between co-detection, elevated serum IP-10 and the pathogenesis of pneumonia.", "These observations suggest an interrelationship between co-detection, elevated serum IP-10 and the pathogenesis of pneumonia. Several limitations of this pilot translational study need to be acknowledged before concluding mixed infection is related to elevated IP-10 and disease severity. Indeed, although viral shedding (e.g., of HRV and HBoV) is common in asymptomatic children, we were unable to evaluate the levels of immunomodulators in the serum samples of a control group.", "Indeed, although viral shedding (e.g., of HRV and HBoV) is common in asymptomatic children, we were unable to evaluate the levels of immunomodulators in the serum samples of a control group. Moreover, although the samples were collected within the first 24 hours after admission, only a single blood sample was processed for each patient. Therefore, a larger, longitudinal study on the etiology and severity of pneumonia will be necessary to confirm these results.", "Therefore, a larger, longitudinal study on the etiology and severity of pneumonia will be necessary to confirm these results. In conclusion, the present findings suggest that mixed respiratory infections and IP-10 may play major, interconnected roles in the pathogenesis of pneumonia. Clinically, assessment and monitoring of induced IP-10 serum level may assist clinicians to improve diagnosis and patient management of severe community-acquired pneumonia.", "Clinically, assessment and monitoring of induced IP-10 serum level may assist clinicians to improve diagnosis and patient management of severe community-acquired pneumonia. Viral and bacterial strains. The 10 ng ml −1 M-CSF (Miltenyi Biotec).", "Viral and bacterial strains. The 10 ng ml −1 M-CSF (Miltenyi Biotec). THP− 1 MDMs were obtained by culturing cells with 10 ng ml -1 phorbol myristate acetate (PMA; Invivogen, Toulouse, France) for 72 hours.", "THP− 1 MDMs were obtained by culturing cells with 10 ng ml -1 phorbol myristate acetate (PMA; Invivogen, Toulouse, France) for 72 hours. Human airway epithelial cells (HAEC, bronchial cell type) originated from a 54-years old woman with no pathology reported (batch number MD056501) were provided by Mucilair (Epithelix, Geneva, Switzerland). Sterility, tissue integrity (TEER), mucus production and cilia beating frequency have been certified by the company.", "Sterility, tissue integrity (TEER), mucus production and cilia beating frequency have been certified by the company. Gene expression profiling. Total cellular mRNA was purified using the RNeasy kit (Qiagen, Hilden, Germany). Reverse-transcription of total mRNA was performed using the RT 2 First Strand Kit (SABiosciences, Hilden, Germany).", "Reverse-transcription of total mRNA was performed using the RT 2 First Strand Kit (SABiosciences, Hilden, Germany). The expression of 84 genes involved in the human innate and adaptive immune responses was evaluated using the RT 2 profiler ™ PCR Array (SABiosciences) according to the manufacturer's recommendations. The Δ Δ Ct method was applied to calculate the fold changes in gene expression for each gene relative to uninfected control cells using the web-based RT 2 profiler PCR Array Data Analysis software (SABiosciences).", "The Δ Δ Ct method was applied to calculate the fold changes in gene expression for each gene relative to uninfected control cells using the web-based RT 2 profiler PCR Array Data Analysis software (SABiosciences). MicroRNA profiling array. Total cellular microRNAs were purified using the miRNeasy Mini kit (Qiagen) and reverse-transcribed using the miScript Reverse Transcription kit (Qiagen).", "Total cellular microRNAs were purified using the miRNeasy Mini kit (Qiagen) and reverse-transcribed using the miScript Reverse Transcription kit (Qiagen). The profiling of 84 miRNAs was performed using the Human Immunopathology miScript miRNA PCR Array kit (Qiagen) according to the manufacturer's instructions. Data were analyzed using the miScript miRNA PCR array data analysis web portal.", "Data were analyzed using the miScript miRNA PCR array data analysis web portal. In silico miRNA target prediction. MiRNA target genes were retrieved and compiled using TargetScan 38 and microRNA.org resource 39 . The interactions between miRNAs and intracellular pathways were predicted using DIANA-miRPath v2.0 40 .", "The interactions between miRNAs and intracellular pathways were predicted using DIANA-miRPath v2.0 40 . THP-1 MDMs were seeded in 24-well plates (0.5 × 10 6 per well) in triplicate, exposed to Influenza A H1N1 (A/Solomon islands/3/2006) virus (IAV) under serum-free conditions for 1 hour and then cultured for 3 hours in fresh RPMI-1640 containing 2% FBS. Streptococcus pneumoniae (SP) serotype 14 was added at 4 hours after IAV infection.", "Streptococcus pneumoniae (SP) serotype 14 was added at 4 hours after IAV infection. Gentamicin (10 μ g ml −1 ) was added 2 hours after SP infection (i.e. 6 hours post-influenza infection) and maintained in the culture media throughout the experiment to kill extracellular bacteria and limit bacterial growth.", "6 hours post-influenza infection) and maintained in the culture media throughout the experiment to kill extracellular bacteria and limit bacterial growth. Cell viability was determined by flow-cytometry using the FITC/Annexin V apoptosis detection kit (BD Biosciences), according to the manufacturer's instructions. #4427975) .", "#4427975) . In this assay, fold changes have been defined by the Δ Δ Ct method using control RNU-44 and -48 as reference microRNAs. Total mRNA was purified from transfected and infected MDMs using the RNeasy kit (Qiagen) and specific primers were used to amplify transforming growth factor beta-2 (TGFB2; F: 5′ -CCATCCCGCCCACTTTCTAC-3′ , R: 5′ -AGCTCAATCCGTTGTTCAGGC-3′ ), SOCS6 (F: 5′ -AAGAATTCATCCCTTGGATTAGGTAAC-3′ , R: 5′ -CAGACTGGAGGTCGTGGAA-3′ ) 41 43 , and 3) absence of wheezing at auscultation, and, 4) first symptoms appearing within the last 14 days, and 5) radiological confirmation of pneumonia as per WHO guidelines 44 .", "Total mRNA was purified from transfected and infected MDMs using the RNeasy kit (Qiagen) and specific primers were used to amplify transforming growth factor beta-2 (TGFB2; F: 5′ -CCATCCCGCCCACTTTCTAC-3′ , R: 5′ -AGCTCAATCCGTTGTTCAGGC-3′ ), SOCS6 (F: 5′ -AAGAATTCATCCCTTGGATTAGGTAAC-3′ , R: 5′ -CAGACTGGAGGTCGTGGAA-3′ ) 41 43 , and 3) absence of wheezing at auscultation, and, 4) first symptoms appearing within the last 14 days, and 5) radiological confirmation of pneumonia as per WHO guidelines 44 . Based on these primary criteria defining pneumonia cases, all 74 cases were retrospectively re-evaluated according to the WHO \"Pocket book of hospital care for children\" 45 criteria to evaluate pneumonia severity. Cases that died during the study, or who had at least one additional clinical signs including central cyanosis, dullness to percussion during chest examination, prostration/lethargy, pleural effusion observed on chest radiography were retrospectively included in the severe pneumonia group.", "Cases that died during the study, or who had at least one additional clinical signs including central cyanosis, dullness to percussion during chest examination, prostration/lethargy, pleural effusion observed on chest radiography were retrospectively included in the severe pneumonia group. Patients without any of these additional clinical signs were included in the non-severe pneumonia group. Table 4 .", "Table 4 . a IP-10 values are expressed in pg ml -1 . IP-10 concentration differences between groups were compared using unpaired Mann-Whitney tests; significant changes (P < 0.05) are in bold. Clinical and molecular analysis. Nasopharyngeal aspirates (NAs) and whole blood samples were collected from children within 24 hours of admission.", "Nasopharyngeal aspirates (NAs) and whole blood samples were collected from children within 24 hours of admission. Whole blood samples were used for complete blood counts, blood culture and multiplex real-time PCR to identify Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae type B 46 . S. pneumoniae serotypes were defined using a 11 multiplex real-time PCR assay targeting the 40 most frequently represented serotypes or serogroups according to protocol developed by Messaoudi et al.", "S. pneumoniae serotypes were defined using a 11 multiplex real-time PCR assay targeting the 40 most frequently represented serotypes or serogroups according to protocol developed by Messaoudi et al. 47 . Serum C-reactive protein (CRP; AssayPro, St. Charles, Missouri, United States) and Procalcitonin (PCT; VIDAS B.R.A.H.M.S; bioMérieux) were quantified from whole-blood samples.", "Serum C-reactive protein (CRP; AssayPro, St. Charles, Missouri, United States) and Procalcitonin (PCT; VIDAS B.R.A.H.M.S; bioMérieux) were quantified from whole-blood samples. Multiplex real-time non quantitative PCR (Fast-Track Diagnostic, Sliema, Malta) was used to detect 19 viruses and five bacteria in the respiratory specimens (NAs and pleural effusions). Mixed detection was defined as 1) PCR-positive for multiple viruses in NAs, 2) positive blood culture or PCR-positive for multiple bacteria in blood or 3) PCR-positive for one or multiple viruses in NAs and one or multiple bacteria in blood (identified by PCR and blood culture).", "Mixed detection was defined as 1) PCR-positive for multiple viruses in NAs, 2) positive blood culture or PCR-positive for multiple bacteria in blood or 3) PCR-positive for one or multiple viruses in NAs and one or multiple bacteria in blood (identified by PCR and blood culture). Ethical approval. The study protocol, informed consent statement, clinical research form, any amendments and all other study documents were submitted to and approved by the Ethical Committee of the Instituto de Investigaciones en Ciencias de la Salud, the Universidad Nacional de Asunción (IICS-UNA) and the Hospital Pediátrico Niños de Acosta Ñu.", "The study protocol, informed consent statement, clinical research form, any amendments and all other study documents were submitted to and approved by the Ethical Committee of the Instituto de Investigaciones en Ciencias de la Salud, the Universidad Nacional de Asunción (IICS-UNA) and the Hospital Pediátrico Niños de Acosta Ñu. Informed consent was obtained from all subjects involved in this study. The clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki.", "The clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki. Statistical analysis. The Chi-square test and Fisher's exact test were used to compare categorical variables; continuous variables and non-normally distributed data were compared using the Mann-Whitney U-test; normally distributed data were compared using unpaired t-tests.", "The Chi-square test and Fisher's exact test were used to compare categorical variables; continuous variables and non-normally distributed data were compared using the Mann-Whitney U-test; normally distributed data were compared using unpaired t-tests. Comparative analyses between experimental conditions (i.e., MOCK, IAV, SP or IAV + SP) were performed using one-way ANOVA with Tukey's post-hoc test or Kruskal-Wallis analysis with Dunn's post-hoc tests. Receiver operating curve (ROC) analysis was used to determine the optimal cut-off value for IP-10 to differentiate between non-severe and severe pneumonia cases.", "Receiver operating curve (ROC) analysis was used to determine the optimal cut-off value for IP-10 to differentiate between non-severe and severe pneumonia cases. P < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism (La Jolla, California, United States)." ]

[ "Mixed viral and bacterial infections are widely described in community-acquired pneumonia; however, the clinical implications of co-infection on the associated immunopathology remain poorly studied. In this study, microRNA, mRNA and cytokine/chemokine secretion profiling were investigated for human monocyte-derived macrophages infected in-vitro with Influenza virus A/H1N1 and/or Streptococcus pneumoniae. We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions.", "We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions. We demonstrated that endogenous miRNA-200a-3p, whose expression was synergistically induced following co-infection, indirectly regulates CXCL10 expression by targeting suppressor of cytokine signaling-6 (SOCS-6), a well-known regulator of the JAK-STAT signaling pathway. Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5 years-old) hospitalized with viral and/or bacterial community-acquired pneumonia.", "Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5 years-old) hospitalized with viral and/or bacterial community-acquired pneumonia. Clinically, among the 74 cases of pneumonia, patients with identified mixed-detection had significantly higher (3.6-fold) serum IP-10 levels than those with a single detection (P = 0.03), and were significantly associated with severe pneumonia (P < 0.01). This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia.", "This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia. Text: Scientific RepoRts | 6:38532 | DOI: 10 .1038/srep38532 pathogenesis of several diseases and has been suggested as a potential biomarker of viral infection 10, 11 , late-onset bacterial infection in premature infants 12 , and a promising biomarker of sepsis and septic shock 13, 14 . Combined analysis of IP-10 and IFN-γ has also been reported as a useful biomarker for diagnosis and monitoring therapeutic efficacy in patients with active tuberculosis [15] [16] [17] , and both remain detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunction 18 .", "Combined analysis of IP-10 and IFN-γ has also been reported as a useful biomarker for diagnosis and monitoring therapeutic efficacy in patients with active tuberculosis [15] [16] [17] , and both remain detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunction 18 . With airway epithelial cells 19 , resident alveolar macrophages (AMs) and blood monocytes-derived macrophages (recruited into tissues under inflammatory conditions 20, 21 ) represent a major line of defense against both pneumococcal (through their high phagocytic capacity [22] [23] [24] ) and influenza infection 25, 26 . So far, no studies have yet focused on the intracellular mechanisms that regulate IP-10 in human blood leukocytes during mixed IAV and SP infection.", "So far, no studies have yet focused on the intracellular mechanisms that regulate IP-10 in human blood leukocytes during mixed IAV and SP infection. Several studies indicated that host non-coding small RNAs (including microRNAs) may function as immunomodulators by regulating several pivotal intracellular processes, such as the innate immune response 27 and antiviral activity 28, 29 ; both of these processes are closely related to toll-like receptor (TLR) signaling pathways. In this study, we firstly investigated the in vitro intracellular mechanisms that mediate the innate immune response in IAV and/or SP infected human monocyte-derived macrophages (MDMs).", "In this study, we firstly investigated the in vitro intracellular mechanisms that mediate the innate immune response in IAV and/or SP infected human monocyte-derived macrophages (MDMs). Using this approach, we observed that mixed-infection of MDMs induces a synergistic production of IP-10 which can be related to a miRNA-200a/JAK-STAT/SOCS-6 regulatory pathway. Subsequently, in a retrospective analysis of clinical samples collected from children ≤ 5 years-old hospitalized with pneumonia, we confirmed that serum IP-10 level could be related to both viral and/or bacterial etiologies and disease severity.", "Subsequently, in a retrospective analysis of clinical samples collected from children ≤ 5 years-old hospitalized with pneumonia, we confirmed that serum IP-10 level could be related to both viral and/or bacterial etiologies and disease severity. Characteristics of MDMs infected by IAV and/or SP. Initially, we investigated in vitro the impact of single and mixed IAV and SP infection on MDMs.", "Initially, we investigated in vitro the impact of single and mixed IAV and SP infection on MDMs. Firstly, active replication of IAV was assessed by qRT-PCR and quantification of new infectious viral particles in the cell supernatants ( Fig. 1a,b ).", "1a,b ). IAV titer increased over time after single infection with IAV and correlated with increased production of negative-strand IAV RNA. Maximum viral replication was observed at 18-24 hours post-infection, after which time both RNA replication and the quantity of infectious particles decreased.", "Maximum viral replication was observed at 18-24 hours post-infection, after which time both RNA replication and the quantity of infectious particles decreased. In this in vitro model, subsequent challenge of IAV-infected MDMs with SP had no significant impact on the production of new infectious viral particles (Fig. 1b) .", "1b) . Together, these results indicate permissive and productive infection of MDMs by IAV. Secondly, we evaluated whether MDMs are permissive for both IAV and SP infection. The presence of pneumococci within IAV-and SP-infected primary MDMs was confirmed at 8 h post-infection (Fig.", "The presence of pneumococci within IAV-and SP-infected primary MDMs was confirmed at 8 h post-infection (Fig. 1c) , suggesting that MDMs are permissive for viral and bacterial co-infection in the early steps of infection. Importantly, confocal co-detection of mixed IAV and SP was only effective following 8 h post-infection due to the bactericidal impact of SP internalization within human macrophages (after 24 h, data not shown).", "Importantly, confocal co-detection of mixed IAV and SP was only effective following 8 h post-infection due to the bactericidal impact of SP internalization within human macrophages (after 24 h, data not shown). Thirdly, we evaluated the impact of single and mixed infection with IAV and SP on MDM viability. Mixed infection significantly decreased cell viability (65.2 ± 4.5% total cell death at 48 hours post-infection; P < 0.0001) compared to single SP and IAV infection (39.6 ± 1.7% and 17.4 ± 1.1% total cell death, respectively; Fig.", "Mixed infection significantly decreased cell viability (65.2 ± 4.5% total cell death at 48 hours post-infection; P < 0.0001) compared to single SP and IAV infection (39.6 ± 1.7% and 17.4 ± 1.1% total cell death, respectively; Fig. 1d ). Taken together, these results confirmed human MDMs are permissive to mixed viral and bacterial infection.", "Taken together, these results confirmed human MDMs are permissive to mixed viral and bacterial infection. mRNA, microRNA and protein expression profiling reveal an overall induction of the host innate immune response following IAV and/or SP infection of MDMs. To investigate the innate immune response orchestrated by IAV-and SP-infected human MDMs, we firstly evaluated the expression of 84 genes involved in the innate and adaptive immune responses (Table S1) ; the major differentially-expressed genes are summarized in Fig.", "To investigate the innate immune response orchestrated by IAV-and SP-infected human MDMs, we firstly evaluated the expression of 84 genes involved in the innate and adaptive immune responses (Table S1) ; the major differentially-expressed genes are summarized in Fig. 2a . Expression profiling indicated an overall induction of genes related to the JAK-STAT, NF-Κ β and TLR signaling pathways.", "Expression profiling indicated an overall induction of genes related to the JAK-STAT, NF-Κ β and TLR signaling pathways. Indeed, all interferon-stimulated genes (ISGs) screened, including CXCL10 (fold-change [FC] = 240.9), CCL-2 (FC = 34.2) and MX-1 (FC = 151.4) were upregulated following mixed infection compared to uninfected cells, most of which are closely related to STAT-1 (FC = 52.3), IRF-7 (FC = 6.8) and IFNB1 (FC = 5.2) also found upregulated in mixed infected cells. Secondly, we investigated the endogenous microRNA expression profiles of IAV-and SP-infected MDMs.", "Secondly, we investigated the endogenous microRNA expression profiles of IAV-and SP-infected MDMs. A selection of microRNAs that were found to be differentially-expressed under different infection conditions are shown in Fig. 2b and Table S2 .", "2b and Table S2 . MiRNA-200a-3p was overexpressed after both single IAV (FC = 6.9), single SP (FC = 3.7) and mixed IAV/SP infection (FC = 7.3), indicating this miRNA may play a role in the innate immune response to viral and bacterial co-infection. Similar miRNA-200a-3p dysregulation profiles were obtained following IAV and/or SP infections of human macrophages-like (THP-1 monocytes-derived macrophages) or primary MDMs (data not shown).", "Similar miRNA-200a-3p dysregulation profiles were obtained following IAV and/or SP infections of human macrophages-like (THP-1 monocytes-derived macrophages) or primary MDMs (data not shown). Thirdly, the secreted levels of various antiviral, pro-inflammatory and immunomodulatory cytokines/chemokines were assayed in IAV-and SP-infected-THP-1 and primary MDM cell supernatants. We observed a remarkable correlation between the mRNA and protein expression profiles of single or mixed infected MDMs especially regarding CXCL-10 and IP-10 expression.", "We observed a remarkable correlation between the mRNA and protein expression profiles of single or mixed infected MDMs especially regarding CXCL-10 and IP-10 expression. Indeed, the level of IP-10 was synergistically increased in the supernatant of IAV-infected THP-1 MDMs exposed to SP (mean: 30,589 ± 16,484 pg ml −1 ) compared to single IAV infection (1,439 ± 566.5 pg ml −1 ) and single SP infection (4,472 ± 2,001 pg ml −1 ; P≤ 0.05; Fig. 2c ) at 24 hours after infection.", "2c ) at 24 hours after infection. In those cells, IP-10 expression reduced over time (48 to 72 hours), coinciding with a significant higher proportion of necrotic and apoptotic cells (Fig. 1d) . The synergistic expression of IP-10 was similarly observed at 24 hours post-infection using primary MDMs (Fig. 2d) .", "2d) . Significantly increased secretion of the other tested cytokines and chemokines was not observed post-infection, even in mixed infected MDMs (Fig. S1 ). Interestingly, a significant production of IP-10 was also observed in supernatants of primary human airway epithelial cells (HAEC) mixed-infected by IAV and SP compared to the single infections (Fig.", "Interestingly, a significant production of IP-10 was also observed in supernatants of primary human airway epithelial cells (HAEC) mixed-infected by IAV and SP compared to the single infections (Fig. 2e) . Taken together, the mRNA and protein profiling results suggested that mixed viral and bacterial infection of MDMs induces a synergistic pro-inflammatory response related to the type-1 interferon and JAK-STAT signaling pathways, with IP-10 as signature of IAV/SP co-infection.", "Taken together, the mRNA and protein profiling results suggested that mixed viral and bacterial infection of MDMs induces a synergistic pro-inflammatory response related to the type-1 interferon and JAK-STAT signaling pathways, with IP-10 as signature of IAV/SP co-infection. Among all microRNAs screened, miR-200a-3p was the most Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 overexpressed in IAV/SP co-infection of human MDMs. In the remainder of this study, we decided to investigate the interconnection between miR-200a-3p expression and the innate immune response.", "In the remainder of this study, we decided to investigate the interconnection between miR-200a-3p expression and the innate immune response. Endogenous miRNA-200a-3p expression correlates with CXCL10 (IP-10) induction following mixed IAV and SP infection of human MDMs. Using a specific Taqman probe assay targeting miR-200a-3p, we confirmed a significant upregulation of miR-200a-3p following mixed IAV and SP infection of human MDMs (Fig.", "Using a specific Taqman probe assay targeting miR-200a-3p, we confirmed a significant upregulation of miR-200a-3p following mixed IAV and SP infection of human MDMs (Fig. 3a) . In this experiment, a more marked up-regulation of miR-200a-3p was observed following IAV+ SP compared to results obtained previously (Fig. 2b) .", "2b) . This discrepancy has been attributed to the use of two different approaches to quantify miR-200a-3p expression. The use of a target-specific stem-loop reverse transcription primer in Fig. 3a allows a better sensitivity of miR-200a-3p detection compared to the non-specific fluorescent dye used in Fig. 2b .", "2b . As the general trend was suggestive of a synergistic induction of miR-200a-3p in response to mixed infection (Fig. 3a) , we hypothesized microRNA-200a-3p may play a role in the regulation of CXCL10 (IP-10), which was also synergistically upregulated in mixed-infected MDMs ( Fig.", "3a) , we hypothesized microRNA-200a-3p may play a role in the regulation of CXCL10 (IP-10), which was also synergistically upregulated in mixed-infected MDMs ( Fig. 2c and d) and primary HAEC ( Statistical analyses were performed using two-way ANOVA with Tukey's post-hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 CXCL10 (Fig.", "Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 CXCL10 (Fig. 3d) . These results suggested miR-200a-3p indirectly regulates CXCL10 and led us to hypothesize that miR-200a-3p controls a potential repressor of the JAK-STAT signaling pathway. . At 18 h after transfection, the MDMs were singly or mixed infected as described previously.", "At 18 h after transfection, the MDMs were singly or mixed infected as described previously. At 8 h post-IAV and/or SP infection, total mRNA was extracted and amplified by PCR using specific primers for the indicated genes. Values represent median ± IQR (a, c) or mean ± SEM (d, e) of three biological replicates.", "Values represent median ± IQR (a, c) or mean ± SEM (d, e) of three biological replicates. Statistical analyses were performed using a Kruskal-Wallis test (non-parametric, one-way ANOVA with Dunn's post-hoc test) for data presented in (a, c). An ordinary two-way ANOVA (with Tukey's post-hoc multiple comparison test) was used for data presented in (d, e).", "An ordinary two-way ANOVA (with Tukey's post-hoc multiple comparison test) was used for data presented in (d, e). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. MiRNA-200a-3p indirectly regulates IP-10 expression by targeting SOCS6.", "MiRNA-200a-3p indirectly regulates IP-10 expression by targeting SOCS6. As shown in Fig. 2a , several JAK-STAT signaling pathway genes were deregulated in mixed IAV-and SP-infected human MDMs; therefore, we hypothesized that miR-200a-3p directly regulates a regulator of the JAK-STAT signaling pathway.", "2a , several JAK-STAT signaling pathway genes were deregulated in mixed IAV-and SP-infected human MDMs; therefore, we hypothesized that miR-200a-3p directly regulates a regulator of the JAK-STAT signaling pathway. Predictive target analysis indicated that the 3' UTR of suppressor of cytokine signaling-6 (SOCS6) may be targeted by miR-200a-3p (Fig. 3b) .", "3b) . SOCS proteins constitute a class of negative regulators of JAK-STAT signaling pathways that are induced by both cytokines and TLR signaling. MiRNA-200a-3p was not predicted to target any of the other six members of the SOCS gene family.", "MiRNA-200a-3p was not predicted to target any of the other six members of the SOCS gene family. Transfection of human MDMs with MIM-200a downregulated SOCS6 (FC = 0.57) while inhibition of miR-200a-3p (INH-200a) upregulated SOCS6 (FC = 1.55), confirming that miR-200a-3p effectively regulates the expression of SOCS6 (Fig. 3e) .", "3e) . Moreover, SOCS6 was synergistically downregulated in IAV-or IAV/SP-infected MDMs overexpressing miRNA-200a (Fig. 3e) , suggesting that both infection and miR-200a-3p negatively regulate the expression of SOCS6. Finally, western blotting confirmed that expression of SOCS-6 sharply reduced following infection, especially after mixed IAV and SP infection (Fig. 3f) .", "3f) . These results indicate miR-200a-3p is strongly induced in response to mixed viral and bacterial co-infection, which in turn leads to downregulation of the JAK-STAT regulator SOCS-6 at both the mRNA and protein levels and subsequent upregulation of IP-10. analyses demonstrated mixed IAV and SP infection of human MDMs and HAEC induced significant production of IP-10.", "analyses demonstrated mixed IAV and SP infection of human MDMs and HAEC induced significant production of IP-10. As blood leukocytes and respiratory tract epithelial cells actively contribute to inflammation during pneumonia, we hypothesized the level of IP-10 in serum of patient with pneumonia may be both indicative of mixed respiratory infection and disease severity. As part of a prospective, hospital-based, multicenter case-control study on the etiology of pneumonia among children under 5-years-old, a total of 74 patients (44 male, 30 female) were included in this pilot evaluation.", "As part of a prospective, hospital-based, multicenter case-control study on the etiology of pneumonia among children under 5-years-old, a total of 74 patients (44 male, 30 female) were included in this pilot evaluation. According to WHO guidelines, retrospective analysis indicated 44 (59.5%) children had clinical signs of non-severe pneumonia and 30 (40.5%) children had signs of severe pneumonia. The main patient characteristics at inclusion are shown in Table 1 .", "The main patient characteristics at inclusion are shown in Table 1 . Patients with severe pneumonia had significant more recorded episodes of dyspnea (P < 0.001), cyanosis (P = 0.03), lower chest indrawing (P < 0.001), dullness to percussion (P < 0.001) and lethargy (P < 0.001) during chest examination than patient with non-severe pneumonia. Moreover, pleural effusions were significantly more observed among critically ill patients and the duration of hospitalization was significantly longer for the children with severe pneumonia than for those with non-severe pneumonia (P = 0.0015).", "Moreover, pleural effusions were significantly more observed among critically ill patients and the duration of hospitalization was significantly longer for the children with severe pneumonia than for those with non-severe pneumonia (P = 0.0015). Two deaths occurred within the group of children retrospectively defined with severe pneumonia. Evaluation of the systemic inflammatory response of the 74 cases is shown in Table 2 .", "Evaluation of the systemic inflammatory response of the 74 cases is shown in Table 2 . Serum level of CRP, IP-10, PCT, G-CSF, IL-6, IL-8 and MIP-1β were significantly more elevated in serum samples from critically ill patients. Patients with severe pneumonia had significantly higher (4.2-fold) serum IP-10 levels than those with a non-severe pneumonia (P < 0.001) suggesting IP-10 as a promising prognostic marker in pneumonia.", "Patients with severe pneumonia had significantly higher (4.2-fold) serum IP-10 levels than those with a non-severe pneumonia (P < 0.001) suggesting IP-10 as a promising prognostic marker in pneumonia. Diagnostic accuracy measures for predicting pneumonia severity using blood-based biomarkers are summarized in Table S3 . Briefly, in this study, the optimal IP-10 cut-off value for identifying patient with severe pneumonia was 4,240 pg ml −1 , with an area under the receiver operating characteristic curve of 0.69 (95% CI, 0.57 to 0.82, P < 0.001).", "Briefly, in this study, the optimal IP-10 cut-off value for identifying patient with severe pneumonia was 4,240 pg ml −1 , with an area under the receiver operating characteristic curve of 0.69 (95% CI, 0.57 to 0.82, P < 0.001). Defining as positive a serum IP-10 level above this cut-off resulted in a sensitivity of 63.3%, specificity of 63.6% and a positive likelihood ratio of 1.74. Prognostic values of IP-10 were closed to procalcitonin (PCT; AUC = 0.70; 95% IC, 0.58 to 0.82, P < 0.001) and IL-6 (AUC = 0.70; 95% IC, 0.58-0.83, P < 0.001).", "Prognostic values of IP-10 were closed to procalcitonin (PCT; AUC = 0.70; 95% IC, 0.58 to 0.82, P < 0.001) and IL-6 (AUC = 0.70; 95% IC, 0.58-0.83, P < 0.001). Multiplex PCR-based screening of respiratory and blood samples reveal a high variety of pathogen associations (Table 3) . Respiratory viruses were detected in the nasal aspirates (NAs) of 63/74 patients (85.1%).", "Respiratory viruses were detected in the nasal aspirates (NAs) of 63/74 patients (85.1%). Etiological bacteria of pneumonia (S. pneumoniae, n = 19; S. aureus, n = 1; or H. influenzae type B, n = 7) were identified via real-time PCR in the blood samples of 27/74 (36.5%) of the patients. Multiplex PCR assays allowed the identification of respiratory bacteria in the blood of 19 patients with negative blood culture results.", "Multiplex PCR assays allowed the identification of respiratory bacteria in the blood of 19 patients with negative blood culture results. Among the 74 cases PCR-positive for respiratory pathogens, a single virus or bacteria were detected in the NAs of 7 (9.4%) and 3 (4.0%) patients, respectively; these 10/74 (13.5%) cases were defined as the single infection group. The mixed infection group included the 62/74 (83.8%) cases in which (1) multiple viruses and/or bacteria were identified in NAs (38/74; 51.3%) without any bacteria identified in blood samples or (2) one or more viruses and/or bacteria were identified in NAs and associated with a blood bacteremia (24/74; 32.4%).", "The mixed infection group included the 62/74 (83.8%) cases in which (1) multiple viruses and/or bacteria were identified in NAs (38/74; 51.3%) without any bacteria identified in blood samples or (2) one or more viruses and/or bacteria were identified in NAs and associated with a blood bacteremia (24/74; 32.4%). We evaluated whether IP-10 serum level could correlate with the viral and bacterial etiologies of pneumonia. Patients with mixed infection had significant higher (3.6-fold) IP-10 serum level than patient with single detection (P = 0.03; Table 4 ).", "Patients with mixed infection had significant higher (3.6-fold) IP-10 serum level than patient with single detection (P = 0.03; Table 4 ). A stratified analysis reveals that the highest IP-10 serum level was observed among patients with both several respiratory pathogens identified (mixed-detection group) and severe pneumonia (14,427 pg ml −1 , IQR (3,981-82,994). In detail, a remarkable IP-10 serum level (142,531 pg ml −1 ), representing 33-fold higher above cut-off value predicting pneumonia severity was observed in patient with hRV in NA co-detected with S. pneumoniae (serotype 14) in pleural effusion and blood.", "In detail, a remarkable IP-10 serum level (142,531 pg ml −1 ), representing 33-fold higher above cut-off value predicting pneumonia severity was observed in patient with hRV in NA co-detected with S. pneumoniae (serotype 14) in pleural effusion and blood. In concordance with our in-vitro model of co-infection, a significant IP-10 level (90,338 pg ml −1 ) was quantified in blood sample of patient with severe bacteremic pneumococcal (serotype 14) pneumonia with a positive co-detection of Influenza B virus in NA. Taken together, these results suggest that high serum IP-10 levels are significantly associated with mixed viral and bacterial detection and also related to pneumonia pathogenesis.", "Taken together, these results suggest that high serum IP-10 levels are significantly associated with mixed viral and bacterial detection and also related to pneumonia pathogenesis. This study provides additional in vitro and clinical data to improve our understanding of the immunopathology of mixed viral and bacterial pneumonia (Fig. 4) .", "4) . The in vitro model of influenza and pneumococcal superinfection of human MDMs demonstrated that mixed infection synergistically induced release of the pro-inflammatory chemokine IP-10, strongly suggesting human Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 blood leukocytes contribute to the immunopathology of pneumonia. Additionally, transcriptomics and omics analyses provided new data on the inflammatory pathways that are activated during mixed infection and related to synergistic induction of the pro-inflammatory chemokine IP-10 in mixed infected cells.", "Additionally, transcriptomics and omics analyses provided new data on the inflammatory pathways that are activated during mixed infection and related to synergistic induction of the pro-inflammatory chemokine IP-10 in mixed infected cells. Our observations are consistent with a recent study describing IP-10 induction as host-proteome signature of both viral and bacterial infections 30 . Of the differentially-expressed genes observed in mixed infected MDMs, the transcription factors STAT-1 and IRF-7 appear to play crucial roles in the regulation of interferon-stimulated genes including CXCL10 (IP-10).", "Of the differentially-expressed genes observed in mixed infected MDMs, the transcription factors STAT-1 and IRF-7 appear to play crucial roles in the regulation of interferon-stimulated genes including CXCL10 (IP-10). By focusing on the intracellular mechanisms that regulate inflammatory pathways, we demonstrated a novel role for miRNA-200a-3p in the regulation of CXCL10 (IP-10). These observations are consistent with previous reports showing that RNA virus infection upregulates miR-155 in macrophages and dendritic cells and also regulates suppressor of cytokine signaling 1 (SOCS1), suggesting the existence of a miRNA/JAK-STAT/SOCS regulatory pathway during viral infection 29 .", "These observations are consistent with previous reports showing that RNA virus infection upregulates miR-155 in macrophages and dendritic cells and also regulates suppressor of cytokine signaling 1 (SOCS1), suggesting the existence of a miRNA/JAK-STAT/SOCS regulatory pathway during viral infection 29 . Our study suggests co-infection leads to overexpression of miR-200a-3p, which in turn targets and downregulates the JAK-STAT regulator SOCS-6 and consequently increases CXCL10 (IP-10) expression. Interestingly, a complementary in-silico approach reveals that several microRNAs that were found dysregulated in our experiments of IAV and SP co-infection of MDMs or HAEC, might target several genes of SOCS family and play similar role than miR-200a-3p.", "Interestingly, a complementary in-silico approach reveals that several microRNAs that were found dysregulated in our experiments of IAV and SP co-infection of MDMs or HAEC, might target several genes of SOCS family and play similar role than miR-200a-3p. Indeed, miRNA-142-3p might target SOCS4, 5, 6 mRNA while miRNA-194-5p might target SOCS2, 3, 4, 5 and 7 mRNA. These observations underline that intra-cellular regulation of IP-10 is not limited to the contribution of a sole microRNA.", "These observations underline that intra-cellular regulation of IP-10 is not limited to the contribution of a sole microRNA. A complex inter-relationship between numerous host microRNAs and inhibitors of the JAK-STAT signaling pathway occur to control host innate inflammatory response against viral and/or bacterial infections. Clinically, the majority of pediatric CAP cases in this study were associated with both positive viral and/or bacterial detection.", "Clinically, the majority of pediatric CAP cases in this study were associated with both positive viral and/or bacterial detection. Respiratory microorganisms were detected in 97% of cases; 51.3% of which were viral-viral, viral-bacterial or bacterial-bacterial co-detected only in nasal aspirates, 32.4% of which co-detected in both nasal aspirates and blood samples. These data are consistent with previous etiological studies of pediatric CAP 3,31-33 .", "These data are consistent with previous etiological studies of pediatric CAP 3,31-33 . S. pneumoniae was the major bacteria identified in blood (19/74; 25.7%) and mainly co-detected with respiratory viruses in NAs (16/19; 84.2%). We observed a very high diversity of viral and bacterial associations in biological samples from children with pneumonia.", "We observed a very high diversity of viral and bacterial associations in biological samples from children with pneumonia. In comparison with IAV and SP14 combination evaluated in-vitro, no pneumonia cases were singly influenza and pneumococcus infected, and no similar co-detection with those two pathogens has been clinically observed. Nevertheless, Influenza B (IVB) virus was identified in 5 patients and two of them had a positive SP co-detection in blood (one non-typable strain and one serotype 14 using our molecular typing test).", "Nevertheless, Influenza B (IVB) virus was identified in 5 patients and two of them had a positive SP co-detection in blood (one non-typable strain and one serotype 14 using our molecular typing test). IVB and SP14 combination seems to be the nearest pathogen co-detection to that in-vitro investigated. Clinically, this co-detection was associated with both a very high IP-10 expression and a very severe pneumonia case definition.", "Clinically, this co-detection was associated with both a very high IP-10 expression and a very severe pneumonia case definition. Interestingly, our translational pilot evaluation reveals IP-10 expression can be induced by several different viral and/or bacterial combinations. As immune response to each pathogen is different, further in-vitro investigations using different pathogens associations are needed to better characterize the mechanisms involved in the immunopathology of pneumonia.", "As immune response to each pathogen is different, further in-vitro investigations using different pathogens associations are needed to better characterize the mechanisms involved in the immunopathology of pneumonia. In this cohort, highest serum IP-10 levels were identified among patients with both several pathogen detected and severe pneumonia, suggesting a significant role of IP-10 on pneumonia pathogenesis. Indeed, high plasma levels of IP-10 have previously been reported in patients with sepsis 12 , and were associated with high mortality rate, especially among patients with CAP 34 .", "Indeed, high plasma levels of IP-10 have previously been reported in patients with sepsis 12 , and were associated with high mortality rate, especially among patients with CAP 34 . Additionally, the IP-10-CXCR3 axis has been related to acute immune lung injury and lymphocyte apoptosis during the development of severe acute respiratory syndrome (SARS) 35, 36 . Moreover, an in vivo study that modeled influenza and pneumococcal superinfection in mice indicated that pro-inflammatory chemokines, including IP-10, play a crucial role in influenza-induced susceptibility to lung neutrophilia, severe immunopathology and mortality 37 .", "Moreover, an in vivo study that modeled influenza and pneumococcal superinfection in mice indicated that pro-inflammatory chemokines, including IP-10, play a crucial role in influenza-induced susceptibility to lung neutrophilia, severe immunopathology and mortality 37 . In this study, markedly elevated IP-10 (92,809 pg ml −1 ) combined with the highest PCT level (74.4 pg ml −1 ) were quantified in the serum sample of a child who died, in whom S. pneumoniae (serotype 9 V) was identified in the blood (PCR and blood culture) and co-detected with Haemophilus influenzae type B in nasal aspirate. These observations suggest an interrelationship between co-detection, elevated serum IP-10 and the pathogenesis of pneumonia.", "These observations suggest an interrelationship between co-detection, elevated serum IP-10 and the pathogenesis of pneumonia. Several limitations of this pilot translational study need to be acknowledged before concluding mixed infection is related to elevated IP-10 and disease severity. Indeed, although viral shedding (e.g., of HRV and HBoV) is common in asymptomatic children, we were unable to evaluate the levels of immunomodulators in the serum samples of a control group.", "Indeed, although viral shedding (e.g., of HRV and HBoV) is common in asymptomatic children, we were unable to evaluate the levels of immunomodulators in the serum samples of a control group. Moreover, although the samples were collected within the first 24 hours after admission, only a single blood sample was processed for each patient. Therefore, a larger, longitudinal study on the etiology and severity of pneumonia will be necessary to confirm these results.", "Therefore, a larger, longitudinal study on the etiology and severity of pneumonia will be necessary to confirm these results. In conclusion, the present findings suggest that mixed respiratory infections and IP-10 may play major, interconnected roles in the pathogenesis of pneumonia. Clinically, assessment and monitoring of induced IP-10 serum level may assist clinicians to improve diagnosis and patient management of severe community-acquired pneumonia.", "Clinically, assessment and monitoring of induced IP-10 serum level may assist clinicians to improve diagnosis and patient management of severe community-acquired pneumonia. Viral and bacterial strains. The 10 ng ml −1 M-CSF (Miltenyi Biotec).", "Viral and bacterial strains. The 10 ng ml −1 M-CSF (Miltenyi Biotec). THP− 1 MDMs were obtained by culturing cells with 10 ng ml -1 phorbol myristate acetate (PMA; Invivogen, Toulouse, France) for 72 hours.", "THP− 1 MDMs were obtained by culturing cells with 10 ng ml -1 phorbol myristate acetate (PMA; Invivogen, Toulouse, France) for 72 hours. Human airway epithelial cells (HAEC, bronchial cell type) originated from a 54-years old woman with no pathology reported (batch number MD056501) were provided by Mucilair (Epithelix, Geneva, Switzerland). Sterility, tissue integrity (TEER), mucus production and cilia beating frequency have been certified by the company.", "Sterility, tissue integrity (TEER), mucus production and cilia beating frequency have been certified by the company. Gene expression profiling. Total cellular mRNA was purified using the RNeasy kit (Qiagen, Hilden, Germany). Reverse-transcription of total mRNA was performed using the RT 2 First Strand Kit (SABiosciences, Hilden, Germany).", "Reverse-transcription of total mRNA was performed using the RT 2 First Strand Kit (SABiosciences, Hilden, Germany). The expression of 84 genes involved in the human innate and adaptive immune responses was evaluated using the RT 2 profiler ™ PCR Array (SABiosciences) according to the manufacturer's recommendations. The Δ Δ Ct method was applied to calculate the fold changes in gene expression for each gene relative to uninfected control cells using the web-based RT 2 profiler PCR Array Data Analysis software (SABiosciences).", "The Δ Δ Ct method was applied to calculate the fold changes in gene expression for each gene relative to uninfected control cells using the web-based RT 2 profiler PCR Array Data Analysis software (SABiosciences). MicroRNA profiling array. Total cellular microRNAs were purified using the miRNeasy Mini kit (Qiagen) and reverse-transcribed using the miScript Reverse Transcription kit (Qiagen).", "Total cellular microRNAs were purified using the miRNeasy Mini kit (Qiagen) and reverse-transcribed using the miScript Reverse Transcription kit (Qiagen). The profiling of 84 miRNAs was performed using the Human Immunopathology miScript miRNA PCR Array kit (Qiagen) according to the manufacturer's instructions. Data were analyzed using the miScript miRNA PCR array data analysis web portal.", "Data were analyzed using the miScript miRNA PCR array data analysis web portal. In silico miRNA target prediction. MiRNA target genes were retrieved and compiled using TargetScan 38 and microRNA.org resource 39 . The interactions between miRNAs and intracellular pathways were predicted using DIANA-miRPath v2.0 40 .", "The interactions between miRNAs and intracellular pathways were predicted using DIANA-miRPath v2.0 40 . THP-1 MDMs were seeded in 24-well plates (0.5 × 10 6 per well) in triplicate, exposed to Influenza A H1N1 (A/Solomon islands/3/2006) virus (IAV) under serum-free conditions for 1 hour and then cultured for 3 hours in fresh RPMI-1640 containing 2% FBS. Streptococcus pneumoniae (SP) serotype 14 was added at 4 hours after IAV infection.", "Streptococcus pneumoniae (SP) serotype 14 was added at 4 hours after IAV infection. Gentamicin (10 μ g ml −1 ) was added 2 hours after SP infection (i.e. 6 hours post-influenza infection) and maintained in the culture media throughout the experiment to kill extracellular bacteria and limit bacterial growth.", "6 hours post-influenza infection) and maintained in the culture media throughout the experiment to kill extracellular bacteria and limit bacterial growth. Cell viability was determined by flow-cytometry using the FITC/Annexin V apoptosis detection kit (BD Biosciences), according to the manufacturer's instructions. #4427975) .", "#4427975) . In this assay, fold changes have been defined by the Δ Δ Ct method using control RNU-44 and -48 as reference microRNAs. Total mRNA was purified from transfected and infected MDMs using the RNeasy kit (Qiagen) and specific primers were used to amplify transforming growth factor beta-2 (TGFB2; F: 5′ -CCATCCCGCCCACTTTCTAC-3′ , R: 5′ -AGCTCAATCCGTTGTTCAGGC-3′ ), SOCS6 (F: 5′ -AAGAATTCATCCCTTGGATTAGGTAAC-3′ , R: 5′ -CAGACTGGAGGTCGTGGAA-3′ ) 41 43 , and 3) absence of wheezing at auscultation, and, 4) first symptoms appearing within the last 14 days, and 5) radiological confirmation of pneumonia as per WHO guidelines 44 .", "Total mRNA was purified from transfected and infected MDMs using the RNeasy kit (Qiagen) and specific primers were used to amplify transforming growth factor beta-2 (TGFB2; F: 5′ -CCATCCCGCCCACTTTCTAC-3′ , R: 5′ -AGCTCAATCCGTTGTTCAGGC-3′ ), SOCS6 (F: 5′ -AAGAATTCATCCCTTGGATTAGGTAAC-3′ , R: 5′ -CAGACTGGAGGTCGTGGAA-3′ ) 41 43 , and 3) absence of wheezing at auscultation, and, 4) first symptoms appearing within the last 14 days, and 5) radiological confirmation of pneumonia as per WHO guidelines 44 . Based on these primary criteria defining pneumonia cases, all 74 cases were retrospectively re-evaluated according to the WHO \"Pocket book of hospital care for children\" 45 criteria to evaluate pneumonia severity. Cases that died during the study, or who had at least one additional clinical signs including central cyanosis, dullness to percussion during chest examination, prostration/lethargy, pleural effusion observed on chest radiography were retrospectively included in the severe pneumonia group.", "Cases that died during the study, or who had at least one additional clinical signs including central cyanosis, dullness to percussion during chest examination, prostration/lethargy, pleural effusion observed on chest radiography were retrospectively included in the severe pneumonia group. Patients without any of these additional clinical signs were included in the non-severe pneumonia group. Table 4 .", "Table 4 . a IP-10 values are expressed in pg ml -1 . IP-10 concentration differences between groups were compared using unpaired Mann-Whitney tests; significant changes (P < 0.05) are in bold. Clinical and molecular analysis. Nasopharyngeal aspirates (NAs) and whole blood samples were collected from children within 24 hours of admission.", "Nasopharyngeal aspirates (NAs) and whole blood samples were collected from children within 24 hours of admission. Whole blood samples were used for complete blood counts, blood culture and multiplex real-time PCR to identify Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae type B 46 . S. pneumoniae serotypes were defined using a 11 multiplex real-time PCR assay targeting the 40 most frequently represented serotypes or serogroups according to protocol developed by Messaoudi et al.", "S. pneumoniae serotypes were defined using a 11 multiplex real-time PCR assay targeting the 40 most frequently represented serotypes or serogroups according to protocol developed by Messaoudi et al. 47 . Serum C-reactive protein (CRP; AssayPro, St. Charles, Missouri, United States) and Procalcitonin (PCT; VIDAS B.R.A.H.M.S; bioMérieux) were quantified from whole-blood samples.", "Serum C-reactive protein (CRP; AssayPro, St. Charles, Missouri, United States) and Procalcitonin (PCT; VIDAS B.R.A.H.M.S; bioMérieux) were quantified from whole-blood samples. Multiplex real-time non quantitative PCR (Fast-Track Diagnostic, Sliema, Malta) was used to detect 19 viruses and five bacteria in the respiratory specimens (NAs and pleural effusions). Mixed detection was defined as 1) PCR-positive for multiple viruses in NAs, 2) positive blood culture or PCR-positive for multiple bacteria in blood or 3) PCR-positive for one or multiple viruses in NAs and one or multiple bacteria in blood (identified by PCR and blood culture).", "Mixed detection was defined as 1) PCR-positive for multiple viruses in NAs, 2) positive blood culture or PCR-positive for multiple bacteria in blood or 3) PCR-positive for one or multiple viruses in NAs and one or multiple bacteria in blood (identified by PCR and blood culture). Ethical approval. The study protocol, informed consent statement, clinical research form, any amendments and all other study documents were submitted to and approved by the Ethical Committee of the Instituto de Investigaciones en Ciencias de la Salud, the Universidad Nacional de Asunción (IICS-UNA) and the Hospital Pediátrico Niños de Acosta Ñu.", "The study protocol, informed consent statement, clinical research form, any amendments and all other study documents were submitted to and approved by the Ethical Committee of the Instituto de Investigaciones en Ciencias de la Salud, the Universidad Nacional de Asunción (IICS-UNA) and the Hospital Pediátrico Niños de Acosta Ñu. Informed consent was obtained from all subjects involved in this study. The clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki.", "The clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki. Statistical analysis. The Chi-square test and Fisher's exact test were used to compare categorical variables; continuous variables and non-normally distributed data were compared using the Mann-Whitney U-test; normally distributed data were compared using unpaired t-tests.", "The Chi-square test and Fisher's exact test were used to compare categorical variables; continuous variables and non-normally distributed data were compared using the Mann-Whitney U-test; normally distributed data were compared using unpaired t-tests. Comparative analyses between experimental conditions (i.e., MOCK, IAV, SP or IAV + SP) were performed using one-way ANOVA with Tukey's post-hoc test or Kruskal-Wallis analysis with Dunn's post-hoc tests. Receiver operating curve (ROC) analysis was used to determine the optimal cut-off value for IP-10 to differentiate between non-severe and severe pneumonia cases.", "Receiver operating curve (ROC) analysis was used to determine the optimal cut-off value for IP-10 to differentiate between non-severe and severe pneumonia cases. P < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism (La Jolla, California, United States)." ]

[ "Mixed viral and bacterial infections are widely described in community-acquired pneumonia; however, the clinical implications of co-infection on the associated immunopathology remain poorly studied. In this study, microRNA, mRNA and cytokine/chemokine secretion profiling were investigated for human monocyte-derived macrophages infected in-vitro with Influenza virus A/H1N1 and/or Streptococcus pneumoniae. We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions.", "We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions. We demonstrated that endogenous miRNA-200a-3p, whose expression was synergistically induced following co-infection, indirectly regulates CXCL10 expression by targeting suppressor of cytokine signaling-6 (SOCS-6), a well-known regulator of the JAK-STAT signaling pathway. Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5 years-old) hospitalized with viral and/or bacterial community-acquired pneumonia.", "Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5 years-old) hospitalized with viral and/or bacterial community-acquired pneumonia. Clinically, among the 74 cases of pneumonia, patients with identified mixed-detection had significantly higher (3.6-fold) serum IP-10 levels than those with a single detection (P = 0.03), and were significantly associated with severe pneumonia (P < 0.01). This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia.", "This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia. Text: Scientific RepoRts | 6:38532 | DOI: 10 .1038/srep38532 pathogenesis of several diseases and has been suggested as a potential biomarker of viral infection 10, 11 , late-onset bacterial infection in premature infants 12 , and a promising biomarker of sepsis and septic shock 13, 14 . Combined analysis of IP-10 and IFN-γ has also been reported as a useful biomarker for diagnosis and monitoring therapeutic efficacy in patients with active tuberculosis [15] [16] [17] , and both remain detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunction 18 .", "Combined analysis of IP-10 and IFN-γ has also been reported as a useful biomarker for diagnosis and monitoring therapeutic efficacy in patients with active tuberculosis [15] [16] [17] , and both remain detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunction 18 . With airway epithelial cells 19 , resident alveolar macrophages (AMs) and blood monocytes-derived macrophages (recruited into tissues under inflammatory conditions 20, 21 ) represent a major line of defense against both pneumococcal (through their high phagocytic capacity [22] [23] [24] ) and influenza infection 25, 26 . So far, no studies have yet focused on the intracellular mechanisms that regulate IP-10 in human blood leukocytes during mixed IAV and SP infection.", "So far, no studies have yet focused on the intracellular mechanisms that regulate IP-10 in human blood leukocytes during mixed IAV and SP infection. Several studies indicated that host non-coding small RNAs (including microRNAs) may function as immunomodulators by regulating several pivotal intracellular processes, such as the innate immune response 27 and antiviral activity 28, 29 ; both of these processes are closely related to toll-like receptor (TLR) signaling pathways. In this study, we firstly investigated the in vitro intracellular mechanisms that mediate the innate immune response in IAV and/or SP infected human monocyte-derived macrophages (MDMs).", "In this study, we firstly investigated the in vitro intracellular mechanisms that mediate the innate immune response in IAV and/or SP infected human monocyte-derived macrophages (MDMs). Using this approach, we observed that mixed-infection of MDMs induces a synergistic production of IP-10 which can be related to a miRNA-200a/JAK-STAT/SOCS-6 regulatory pathway. Subsequently, in a retrospective analysis of clinical samples collected from children ≤ 5 years-old hospitalized with pneumonia, we confirmed that serum IP-10 level could be related to both viral and/or bacterial etiologies and disease severity.", "Subsequently, in a retrospective analysis of clinical samples collected from children ≤ 5 years-old hospitalized with pneumonia, we confirmed that serum IP-10 level could be related to both viral and/or bacterial etiologies and disease severity. Characteristics of MDMs infected by IAV and/or SP. Initially, we investigated in vitro the impact of single and mixed IAV and SP infection on MDMs.", "Initially, we investigated in vitro the impact of single and mixed IAV and SP infection on MDMs. Firstly, active replication of IAV was assessed by qRT-PCR and quantification of new infectious viral particles in the cell supernatants ( Fig. 1a,b ).", "1a,b ). IAV titer increased over time after single infection with IAV and correlated with increased production of negative-strand IAV RNA. Maximum viral replication was observed at 18-24 hours post-infection, after which time both RNA replication and the quantity of infectious particles decreased.", "Maximum viral replication was observed at 18-24 hours post-infection, after which time both RNA replication and the quantity of infectious particles decreased. In this in vitro model, subsequent challenge of IAV-infected MDMs with SP had no significant impact on the production of new infectious viral particles (Fig. 1b) .", "1b) . Together, these results indicate permissive and productive infection of MDMs by IAV. Secondly, we evaluated whether MDMs are permissive for both IAV and SP infection. The presence of pneumococci within IAV-and SP-infected primary MDMs was confirmed at 8 h post-infection (Fig.", "The presence of pneumococci within IAV-and SP-infected primary MDMs was confirmed at 8 h post-infection (Fig. 1c) , suggesting that MDMs are permissive for viral and bacterial co-infection in the early steps of infection. Importantly, confocal co-detection of mixed IAV and SP was only effective following 8 h post-infection due to the bactericidal impact of SP internalization within human macrophages (after 24 h, data not shown).", "Importantly, confocal co-detection of mixed IAV and SP was only effective following 8 h post-infection due to the bactericidal impact of SP internalization within human macrophages (after 24 h, data not shown). Thirdly, we evaluated the impact of single and mixed infection with IAV and SP on MDM viability. Mixed infection significantly decreased cell viability (65.2 ± 4.5% total cell death at 48 hours post-infection; P < 0.0001) compared to single SP and IAV infection (39.6 ± 1.7% and 17.4 ± 1.1% total cell death, respectively; Fig.", "Mixed infection significantly decreased cell viability (65.2 ± 4.5% total cell death at 48 hours post-infection; P < 0.0001) compared to single SP and IAV infection (39.6 ± 1.7% and 17.4 ± 1.1% total cell death, respectively; Fig. 1d ). Taken together, these results confirmed human MDMs are permissive to mixed viral and bacterial infection.", "Taken together, these results confirmed human MDMs are permissive to mixed viral and bacterial infection. mRNA, microRNA and protein expression profiling reveal an overall induction of the host innate immune response following IAV and/or SP infection of MDMs. To investigate the innate immune response orchestrated by IAV-and SP-infected human MDMs, we firstly evaluated the expression of 84 genes involved in the innate and adaptive immune responses (Table S1) ; the major differentially-expressed genes are summarized in Fig.", "To investigate the innate immune response orchestrated by IAV-and SP-infected human MDMs, we firstly evaluated the expression of 84 genes involved in the innate and adaptive immune responses (Table S1) ; the major differentially-expressed genes are summarized in Fig. 2a . Expression profiling indicated an overall induction of genes related to the JAK-STAT, NF-Κ β and TLR signaling pathways.", "Expression profiling indicated an overall induction of genes related to the JAK-STAT, NF-Κ β and TLR signaling pathways. Indeed, all interferon-stimulated genes (ISGs) screened, including CXCL10 (fold-change [FC] = 240.9), CCL-2 (FC = 34.2) and MX-1 (FC = 151.4) were upregulated following mixed infection compared to uninfected cells, most of which are closely related to STAT-1 (FC = 52.3), IRF-7 (FC = 6.8) and IFNB1 (FC = 5.2) also found upregulated in mixed infected cells. Secondly, we investigated the endogenous microRNA expression profiles of IAV-and SP-infected MDMs.", "Secondly, we investigated the endogenous microRNA expression profiles of IAV-and SP-infected MDMs. A selection of microRNAs that were found to be differentially-expressed under different infection conditions are shown in Fig. 2b and Table S2 .", "2b and Table S2 . MiRNA-200a-3p was overexpressed after both single IAV (FC = 6.9), single SP (FC = 3.7) and mixed IAV/SP infection (FC = 7.3), indicating this miRNA may play a role in the innate immune response to viral and bacterial co-infection. Similar miRNA-200a-3p dysregulation profiles were obtained following IAV and/or SP infections of human macrophages-like (THP-1 monocytes-derived macrophages) or primary MDMs (data not shown).", "Similar miRNA-200a-3p dysregulation profiles were obtained following IAV and/or SP infections of human macrophages-like (THP-1 monocytes-derived macrophages) or primary MDMs (data not shown). Thirdly, the secreted levels of various antiviral, pro-inflammatory and immunomodulatory cytokines/chemokines were assayed in IAV-and SP-infected-THP-1 and primary MDM cell supernatants. We observed a remarkable correlation between the mRNA and protein expression profiles of single or mixed infected MDMs especially regarding CXCL-10 and IP-10 expression.", "We observed a remarkable correlation between the mRNA and protein expression profiles of single or mixed infected MDMs especially regarding CXCL-10 and IP-10 expression. Indeed, the level of IP-10 was synergistically increased in the supernatant of IAV-infected THP-1 MDMs exposed to SP (mean: 30,589 ± 16,484 pg ml −1 ) compared to single IAV infection (1,439 ± 566.5 pg ml −1 ) and single SP infection (4,472 ± 2,001 pg ml −1 ; P≤ 0.05; Fig. 2c ) at 24 hours after infection.", "2c ) at 24 hours after infection. In those cells, IP-10 expression reduced over time (48 to 72 hours), coinciding with a significant higher proportion of necrotic and apoptotic cells (Fig. 1d) . The synergistic expression of IP-10 was similarly observed at 24 hours post-infection using primary MDMs (Fig. 2d) .", "2d) . Significantly increased secretion of the other tested cytokines and chemokines was not observed post-infection, even in mixed infected MDMs (Fig. S1 ). Interestingly, a significant production of IP-10 was also observed in supernatants of primary human airway epithelial cells (HAEC) mixed-infected by IAV and SP compared to the single infections (Fig.", "Interestingly, a significant production of IP-10 was also observed in supernatants of primary human airway epithelial cells (HAEC) mixed-infected by IAV and SP compared to the single infections (Fig. 2e) . Taken together, the mRNA and protein profiling results suggested that mixed viral and bacterial infection of MDMs induces a synergistic pro-inflammatory response related to the type-1 interferon and JAK-STAT signaling pathways, with IP-10 as signature of IAV/SP co-infection.", "Taken together, the mRNA and protein profiling results suggested that mixed viral and bacterial infection of MDMs induces a synergistic pro-inflammatory response related to the type-1 interferon and JAK-STAT signaling pathways, with IP-10 as signature of IAV/SP co-infection. Among all microRNAs screened, miR-200a-3p was the most Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 overexpressed in IAV/SP co-infection of human MDMs. In the remainder of this study, we decided to investigate the interconnection between miR-200a-3p expression and the innate immune response.", "In the remainder of this study, we decided to investigate the interconnection between miR-200a-3p expression and the innate immune response. Endogenous miRNA-200a-3p expression correlates with CXCL10 (IP-10) induction following mixed IAV and SP infection of human MDMs. Using a specific Taqman probe assay targeting miR-200a-3p, we confirmed a significant upregulation of miR-200a-3p following mixed IAV and SP infection of human MDMs (Fig.", "Using a specific Taqman probe assay targeting miR-200a-3p, we confirmed a significant upregulation of miR-200a-3p following mixed IAV and SP infection of human MDMs (Fig. 3a) . In this experiment, a more marked up-regulation of miR-200a-3p was observed following IAV+ SP compared to results obtained previously (Fig. 2b) .", "2b) . This discrepancy has been attributed to the use of two different approaches to quantify miR-200a-3p expression. The use of a target-specific stem-loop reverse transcription primer in Fig. 3a allows a better sensitivity of miR-200a-3p detection compared to the non-specific fluorescent dye used in Fig. 2b .", "2b . As the general trend was suggestive of a synergistic induction of miR-200a-3p in response to mixed infection (Fig. 3a) , we hypothesized microRNA-200a-3p may play a role in the regulation of CXCL10 (IP-10), which was also synergistically upregulated in mixed-infected MDMs ( Fig.", "3a) , we hypothesized microRNA-200a-3p may play a role in the regulation of CXCL10 (IP-10), which was also synergistically upregulated in mixed-infected MDMs ( Fig. 2c and d) and primary HAEC ( Statistical analyses were performed using two-way ANOVA with Tukey's post-hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 CXCL10 (Fig.", "Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 CXCL10 (Fig. 3d) . These results suggested miR-200a-3p indirectly regulates CXCL10 and led us to hypothesize that miR-200a-3p controls a potential repressor of the JAK-STAT signaling pathway. . At 18 h after transfection, the MDMs were singly or mixed infected as described previously.", "At 18 h after transfection, the MDMs were singly or mixed infected as described previously. At 8 h post-IAV and/or SP infection, total mRNA was extracted and amplified by PCR using specific primers for the indicated genes. Values represent median ± IQR (a, c) or mean ± SEM (d, e) of three biological replicates.", "Values represent median ± IQR (a, c) or mean ± SEM (d, e) of three biological replicates. Statistical analyses were performed using a Kruskal-Wallis test (non-parametric, one-way ANOVA with Dunn's post-hoc test) for data presented in (a, c). An ordinary two-way ANOVA (with Tukey's post-hoc multiple comparison test) was used for data presented in (d, e).", "An ordinary two-way ANOVA (with Tukey's post-hoc multiple comparison test) was used for data presented in (d, e). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. MiRNA-200a-3p indirectly regulates IP-10 expression by targeting SOCS6.", "MiRNA-200a-3p indirectly regulates IP-10 expression by targeting SOCS6. As shown in Fig. 2a , several JAK-STAT signaling pathway genes were deregulated in mixed IAV-and SP-infected human MDMs; therefore, we hypothesized that miR-200a-3p directly regulates a regulator of the JAK-STAT signaling pathway.", "2a , several JAK-STAT signaling pathway genes were deregulated in mixed IAV-and SP-infected human MDMs; therefore, we hypothesized that miR-200a-3p directly regulates a regulator of the JAK-STAT signaling pathway. Predictive target analysis indicated that the 3' UTR of suppressor of cytokine signaling-6 (SOCS6) may be targeted by miR-200a-3p (Fig. 3b) .", "3b) . SOCS proteins constitute a class of negative regulators of JAK-STAT signaling pathways that are induced by both cytokines and TLR signaling. MiRNA-200a-3p was not predicted to target any of the other six members of the SOCS gene family.", "MiRNA-200a-3p was not predicted to target any of the other six members of the SOCS gene family. Transfection of human MDMs with MIM-200a downregulated SOCS6 (FC = 0.57) while inhibition of miR-200a-3p (INH-200a) upregulated SOCS6 (FC = 1.55), confirming that miR-200a-3p effectively regulates the expression of SOCS6 (Fig. 3e) .", "3e) . Moreover, SOCS6 was synergistically downregulated in IAV-or IAV/SP-infected MDMs overexpressing miRNA-200a (Fig. 3e) , suggesting that both infection and miR-200a-3p negatively regulate the expression of SOCS6. Finally, western blotting confirmed that expression of SOCS-6 sharply reduced following infection, especially after mixed IAV and SP infection (Fig. 3f) .", "3f) . These results indicate miR-200a-3p is strongly induced in response to mixed viral and bacterial co-infection, which in turn leads to downregulation of the JAK-STAT regulator SOCS-6 at both the mRNA and protein levels and subsequent upregulation of IP-10. analyses demonstrated mixed IAV and SP infection of human MDMs and HAEC induced significant production of IP-10.", "analyses demonstrated mixed IAV and SP infection of human MDMs and HAEC induced significant production of IP-10. As blood leukocytes and respiratory tract epithelial cells actively contribute to inflammation during pneumonia, we hypothesized the level of IP-10 in serum of patient with pneumonia may be both indicative of mixed respiratory infection and disease severity. As part of a prospective, hospital-based, multicenter case-control study on the etiology of pneumonia among children under 5-years-old, a total of 74 patients (44 male, 30 female) were included in this pilot evaluation.", "As part of a prospective, hospital-based, multicenter case-control study on the etiology of pneumonia among children under 5-years-old, a total of 74 patients (44 male, 30 female) were included in this pilot evaluation. According to WHO guidelines, retrospective analysis indicated 44 (59.5%) children had clinical signs of non-severe pneumonia and 30 (40.5%) children had signs of severe pneumonia. The main patient characteristics at inclusion are shown in Table 1 .", "The main patient characteristics at inclusion are shown in Table 1 . Patients with severe pneumonia had significant more recorded episodes of dyspnea (P < 0.001), cyanosis (P = 0.03), lower chest indrawing (P < 0.001), dullness to percussion (P < 0.001) and lethargy (P < 0.001) during chest examination than patient with non-severe pneumonia. Moreover, pleural effusions were significantly more observed among critically ill patients and the duration of hospitalization was significantly longer for the children with severe pneumonia than for those with non-severe pneumonia (P = 0.0015).", "Moreover, pleural effusions were significantly more observed among critically ill patients and the duration of hospitalization was significantly longer for the children with severe pneumonia than for those with non-severe pneumonia (P = 0.0015). Two deaths occurred within the group of children retrospectively defined with severe pneumonia. Evaluation of the systemic inflammatory response of the 74 cases is shown in Table 2 .", "Evaluation of the systemic inflammatory response of the 74 cases is shown in Table 2 . Serum level of CRP, IP-10, PCT, G-CSF, IL-6, IL-8 and MIP-1β were significantly more elevated in serum samples from critically ill patients. Patients with severe pneumonia had significantly higher (4.2-fold) serum IP-10 levels than those with a non-severe pneumonia (P < 0.001) suggesting IP-10 as a promising prognostic marker in pneumonia.", "Patients with severe pneumonia had significantly higher (4.2-fold) serum IP-10 levels than those with a non-severe pneumonia (P < 0.001) suggesting IP-10 as a promising prognostic marker in pneumonia. Diagnostic accuracy measures for predicting pneumonia severity using blood-based biomarkers are summarized in Table S3 . Briefly, in this study, the optimal IP-10 cut-off value for identifying patient with severe pneumonia was 4,240 pg ml −1 , with an area under the receiver operating characteristic curve of 0.69 (95% CI, 0.57 to 0.82, P < 0.001).", "Briefly, in this study, the optimal IP-10 cut-off value for identifying patient with severe pneumonia was 4,240 pg ml −1 , with an area under the receiver operating characteristic curve of 0.69 (95% CI, 0.57 to 0.82, P < 0.001). Defining as positive a serum IP-10 level above this cut-off resulted in a sensitivity of 63.3%, specificity of 63.6% and a positive likelihood ratio of 1.74. Prognostic values of IP-10 were closed to procalcitonin (PCT; AUC = 0.70; 95% IC, 0.58 to 0.82, P < 0.001) and IL-6 (AUC = 0.70; 95% IC, 0.58-0.83, P < 0.001).", "Prognostic values of IP-10 were closed to procalcitonin (PCT; AUC = 0.70; 95% IC, 0.58 to 0.82, P < 0.001) and IL-6 (AUC = 0.70; 95% IC, 0.58-0.83, P < 0.001). Multiplex PCR-based screening of respiratory and blood samples reveal a high variety of pathogen associations (Table 3) . Respiratory viruses were detected in the nasal aspirates (NAs) of 63/74 patients (85.1%).", "Respiratory viruses were detected in the nasal aspirates (NAs) of 63/74 patients (85.1%). Etiological bacteria of pneumonia (S. pneumoniae, n = 19; S. aureus, n = 1; or H. influenzae type B, n = 7) were identified via real-time PCR in the blood samples of 27/74 (36.5%) of the patients. Multiplex PCR assays allowed the identification of respiratory bacteria in the blood of 19 patients with negative blood culture results.", "Multiplex PCR assays allowed the identification of respiratory bacteria in the blood of 19 patients with negative blood culture results. Among the 74 cases PCR-positive for respiratory pathogens, a single virus or bacteria were detected in the NAs of 7 (9.4%) and 3 (4.0%) patients, respectively; these 10/74 (13.5%) cases were defined as the single infection group. The mixed infection group included the 62/74 (83.8%) cases in which (1) multiple viruses and/or bacteria were identified in NAs (38/74; 51.3%) without any bacteria identified in blood samples or (2) one or more viruses and/or bacteria were identified in NAs and associated with a blood bacteremia (24/74; 32.4%).", "The mixed infection group included the 62/74 (83.8%) cases in which (1) multiple viruses and/or bacteria were identified in NAs (38/74; 51.3%) without any bacteria identified in blood samples or (2) one or more viruses and/or bacteria were identified in NAs and associated with a blood bacteremia (24/74; 32.4%). We evaluated whether IP-10 serum level could correlate with the viral and bacterial etiologies of pneumonia. Patients with mixed infection had significant higher (3.6-fold) IP-10 serum level than patient with single detection (P = 0.03; Table 4 ).", "Patients with mixed infection had significant higher (3.6-fold) IP-10 serum level than patient with single detection (P = 0.03; Table 4 ). A stratified analysis reveals that the highest IP-10 serum level was observed among patients with both several respiratory pathogens identified (mixed-detection group) and severe pneumonia (14,427 pg ml −1 , IQR (3,981-82,994). In detail, a remarkable IP-10 serum level (142,531 pg ml −1 ), representing 33-fold higher above cut-off value predicting pneumonia severity was observed in patient with hRV in NA co-detected with S. pneumoniae (serotype 14) in pleural effusion and blood.", "In detail, a remarkable IP-10 serum level (142,531 pg ml −1 ), representing 33-fold higher above cut-off value predicting pneumonia severity was observed in patient with hRV in NA co-detected with S. pneumoniae (serotype 14) in pleural effusion and blood. In concordance with our in-vitro model of co-infection, a significant IP-10 level (90,338 pg ml −1 ) was quantified in blood sample of patient with severe bacteremic pneumococcal (serotype 14) pneumonia with a positive co-detection of Influenza B virus in NA. Taken together, these results suggest that high serum IP-10 levels are significantly associated with mixed viral and bacterial detection and also related to pneumonia pathogenesis.", "Taken together, these results suggest that high serum IP-10 levels are significantly associated with mixed viral and bacterial detection and also related to pneumonia pathogenesis. This study provides additional in vitro and clinical data to improve our understanding of the immunopathology of mixed viral and bacterial pneumonia (Fig. 4) .", "4) . The in vitro model of influenza and pneumococcal superinfection of human MDMs demonstrated that mixed infection synergistically induced release of the pro-inflammatory chemokine IP-10, strongly suggesting human Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 blood leukocytes contribute to the immunopathology of pneumonia. Additionally, transcriptomics and omics analyses provided new data on the inflammatory pathways that are activated during mixed infection and related to synergistic induction of the pro-inflammatory chemokine IP-10 in mixed infected cells.", "Additionally, transcriptomics and omics analyses provided new data on the inflammatory pathways that are activated during mixed infection and related to synergistic induction of the pro-inflammatory chemokine IP-10 in mixed infected cells. Our observations are consistent with a recent study describing IP-10 induction as host-proteome signature of both viral and bacterial infections 30 . Of the differentially-expressed genes observed in mixed infected MDMs, the transcription factors STAT-1 and IRF-7 appear to play crucial roles in the regulation of interferon-stimulated genes including CXCL10 (IP-10).", "Of the differentially-expressed genes observed in mixed infected MDMs, the transcription factors STAT-1 and IRF-7 appear to play crucial roles in the regulation of interferon-stimulated genes including CXCL10 (IP-10). By focusing on the intracellular mechanisms that regulate inflammatory pathways, we demonstrated a novel role for miRNA-200a-3p in the regulation of CXCL10 (IP-10). These observations are consistent with previous reports showing that RNA virus infection upregulates miR-155 in macrophages and dendritic cells and also regulates suppressor of cytokine signaling 1 (SOCS1), suggesting the existence of a miRNA/JAK-STAT/SOCS regulatory pathway during viral infection 29 .", "These observations are consistent with previous reports showing that RNA virus infection upregulates miR-155 in macrophages and dendritic cells and also regulates suppressor of cytokine signaling 1 (SOCS1), suggesting the existence of a miRNA/JAK-STAT/SOCS regulatory pathway during viral infection 29 . Our study suggests co-infection leads to overexpression of miR-200a-3p, which in turn targets and downregulates the JAK-STAT regulator SOCS-6 and consequently increases CXCL10 (IP-10) expression. Interestingly, a complementary in-silico approach reveals that several microRNAs that were found dysregulated in our experiments of IAV and SP co-infection of MDMs or HAEC, might target several genes of SOCS family and play similar role than miR-200a-3p.", "Interestingly, a complementary in-silico approach reveals that several microRNAs that were found dysregulated in our experiments of IAV and SP co-infection of MDMs or HAEC, might target several genes of SOCS family and play similar role than miR-200a-3p. Indeed, miRNA-142-3p might target SOCS4, 5, 6 mRNA while miRNA-194-5p might target SOCS2, 3, 4, 5 and 7 mRNA. These observations underline that intra-cellular regulation of IP-10 is not limited to the contribution of a sole microRNA.", "These observations underline that intra-cellular regulation of IP-10 is not limited to the contribution of a sole microRNA. A complex inter-relationship between numerous host microRNAs and inhibitors of the JAK-STAT signaling pathway occur to control host innate inflammatory response against viral and/or bacterial infections. Clinically, the majority of pediatric CAP cases in this study were associated with both positive viral and/or bacterial detection.", "Clinically, the majority of pediatric CAP cases in this study were associated with both positive viral and/or bacterial detection. Respiratory microorganisms were detected in 97% of cases; 51.3% of which were viral-viral, viral-bacterial or bacterial-bacterial co-detected only in nasal aspirates, 32.4% of which co-detected in both nasal aspirates and blood samples. These data are consistent with previous etiological studies of pediatric CAP 3,31-33 .", "These data are consistent with previous etiological studies of pediatric CAP 3,31-33 . S. pneumoniae was the major bacteria identified in blood (19/74; 25.7%) and mainly co-detected with respiratory viruses in NAs (16/19; 84.2%). We observed a very high diversity of viral and bacterial associations in biological samples from children with pneumonia.", "We observed a very high diversity of viral and bacterial associations in biological samples from children with pneumonia. In comparison with IAV and SP14 combination evaluated in-vitro, no pneumonia cases were singly influenza and pneumococcus infected, and no similar co-detection with those two pathogens has been clinically observed. Nevertheless, Influenza B (IVB) virus was identified in 5 patients and two of them had a positive SP co-detection in blood (one non-typable strain and one serotype 14 using our molecular typing test).", "Nevertheless, Influenza B (IVB) virus was identified in 5 patients and two of them had a positive SP co-detection in blood (one non-typable strain and one serotype 14 using our molecular typing test). IVB and SP14 combination seems to be the nearest pathogen co-detection to that in-vitro investigated. Clinically, this co-detection was associated with both a very high IP-10 expression and a very severe pneumonia case definition.", "Clinically, this co-detection was associated with both a very high IP-10 expression and a very severe pneumonia case definition. Interestingly, our translational pilot evaluation reveals IP-10 expression can be induced by several different viral and/or bacterial combinations. As immune response to each pathogen is different, further in-vitro investigations using different pathogens associations are needed to better characterize the mechanisms involved in the immunopathology of pneumonia.", "As immune response to each pathogen is different, further in-vitro investigations using different pathogens associations are needed to better characterize the mechanisms involved in the immunopathology of pneumonia. In this cohort, highest serum IP-10 levels were identified among patients with both several pathogen detected and severe pneumonia, suggesting a significant role of IP-10 on pneumonia pathogenesis. Indeed, high plasma levels of IP-10 have previously been reported in patients with sepsis 12 , and were associated with high mortality rate, especially among patients with CAP 34 .", "Indeed, high plasma levels of IP-10 have previously been reported in patients with sepsis 12 , and were associated with high mortality rate, especially among patients with CAP 34 . Additionally, the IP-10-CXCR3 axis has been related to acute immune lung injury and lymphocyte apoptosis during the development of severe acute respiratory syndrome (SARS) 35, 36 . Moreover, an in vivo study that modeled influenza and pneumococcal superinfection in mice indicated that pro-inflammatory chemokines, including IP-10, play a crucial role in influenza-induced susceptibility to lung neutrophilia, severe immunopathology and mortality 37 .", "Moreover, an in vivo study that modeled influenza and pneumococcal superinfection in mice indicated that pro-inflammatory chemokines, including IP-10, play a crucial role in influenza-induced susceptibility to lung neutrophilia, severe immunopathology and mortality 37 . In this study, markedly elevated IP-10 (92,809 pg ml −1 ) combined with the highest PCT level (74.4 pg ml −1 ) were quantified in the serum sample of a child who died, in whom S. pneumoniae (serotype 9 V) was identified in the blood (PCR and blood culture) and co-detected with Haemophilus influenzae type B in nasal aspirate. These observations suggest an interrelationship between co-detection, elevated serum IP-10 and the pathogenesis of pneumonia.", "These observations suggest an interrelationship between co-detection, elevated serum IP-10 and the pathogenesis of pneumonia. Several limitations of this pilot translational study need to be acknowledged before concluding mixed infection is related to elevated IP-10 and disease severity. Indeed, although viral shedding (e.g., of HRV and HBoV) is common in asymptomatic children, we were unable to evaluate the levels of immunomodulators in the serum samples of a control group.", "Indeed, although viral shedding (e.g., of HRV and HBoV) is common in asymptomatic children, we were unable to evaluate the levels of immunomodulators in the serum samples of a control group. Moreover, although the samples were collected within the first 24 hours after admission, only a single blood sample was processed for each patient. Therefore, a larger, longitudinal study on the etiology and severity of pneumonia will be necessary to confirm these results.", "Therefore, a larger, longitudinal study on the etiology and severity of pneumonia will be necessary to confirm these results. In conclusion, the present findings suggest that mixed respiratory infections and IP-10 may play major, interconnected roles in the pathogenesis of pneumonia. Clinically, assessment and monitoring of induced IP-10 serum level may assist clinicians to improve diagnosis and patient management of severe community-acquired pneumonia.", "Clinically, assessment and monitoring of induced IP-10 serum level may assist clinicians to improve diagnosis and patient management of severe community-acquired pneumonia. Viral and bacterial strains. The 10 ng ml −1 M-CSF (Miltenyi Biotec).", "Viral and bacterial strains. The 10 ng ml −1 M-CSF (Miltenyi Biotec). THP− 1 MDMs were obtained by culturing cells with 10 ng ml -1 phorbol myristate acetate (PMA; Invivogen, Toulouse, France) for 72 hours.", "THP− 1 MDMs were obtained by culturing cells with 10 ng ml -1 phorbol myristate acetate (PMA; Invivogen, Toulouse, France) for 72 hours. Human airway epithelial cells (HAEC, bronchial cell type) originated from a 54-years old woman with no pathology reported (batch number MD056501) were provided by Mucilair (Epithelix, Geneva, Switzerland). Sterility, tissue integrity (TEER), mucus production and cilia beating frequency have been certified by the company.", "Sterility, tissue integrity (TEER), mucus production and cilia beating frequency have been certified by the company. Gene expression profiling. Total cellular mRNA was purified using the RNeasy kit (Qiagen, Hilden, Germany). Reverse-transcription of total mRNA was performed using the RT 2 First Strand Kit (SABiosciences, Hilden, Germany).", "Reverse-transcription of total mRNA was performed using the RT 2 First Strand Kit (SABiosciences, Hilden, Germany). The expression of 84 genes involved in the human innate and adaptive immune responses was evaluated using the RT 2 profiler ™ PCR Array (SABiosciences) according to the manufacturer's recommendations. The Δ Δ Ct method was applied to calculate the fold changes in gene expression for each gene relative to uninfected control cells using the web-based RT 2 profiler PCR Array Data Analysis software (SABiosciences).", "The Δ Δ Ct method was applied to calculate the fold changes in gene expression for each gene relative to uninfected control cells using the web-based RT 2 profiler PCR Array Data Analysis software (SABiosciences). MicroRNA profiling array. Total cellular microRNAs were purified using the miRNeasy Mini kit (Qiagen) and reverse-transcribed using the miScript Reverse Transcription kit (Qiagen).", "Total cellular microRNAs were purified using the miRNeasy Mini kit (Qiagen) and reverse-transcribed using the miScript Reverse Transcription kit (Qiagen). The profiling of 84 miRNAs was performed using the Human Immunopathology miScript miRNA PCR Array kit (Qiagen) according to the manufacturer's instructions. Data were analyzed using the miScript miRNA PCR array data analysis web portal.", "Data were analyzed using the miScript miRNA PCR array data analysis web portal. In silico miRNA target prediction. MiRNA target genes were retrieved and compiled using TargetScan 38 and microRNA.org resource 39 . The interactions between miRNAs and intracellular pathways were predicted using DIANA-miRPath v2.0 40 .", "The interactions between miRNAs and intracellular pathways were predicted using DIANA-miRPath v2.0 40 . THP-1 MDMs were seeded in 24-well plates (0.5 × 10 6 per well) in triplicate, exposed to Influenza A H1N1 (A/Solomon islands/3/2006) virus (IAV) under serum-free conditions for 1 hour and then cultured for 3 hours in fresh RPMI-1640 containing 2% FBS. Streptococcus pneumoniae (SP) serotype 14 was added at 4 hours after IAV infection.", "Streptococcus pneumoniae (SP) serotype 14 was added at 4 hours after IAV infection. Gentamicin (10 μ g ml −1 ) was added 2 hours after SP infection (i.e. 6 hours post-influenza infection) and maintained in the culture media throughout the experiment to kill extracellular bacteria and limit bacterial growth.", "6 hours post-influenza infection) and maintained in the culture media throughout the experiment to kill extracellular bacteria and limit bacterial growth. Cell viability was determined by flow-cytometry using the FITC/Annexin V apoptosis detection kit (BD Biosciences), according to the manufacturer's instructions. #4427975) .", "#4427975) . In this assay, fold changes have been defined by the Δ Δ Ct method using control RNU-44 and -48 as reference microRNAs. Total mRNA was purified from transfected and infected MDMs using the RNeasy kit (Qiagen) and specific primers were used to amplify transforming growth factor beta-2 (TGFB2; F: 5′ -CCATCCCGCCCACTTTCTAC-3′ , R: 5′ -AGCTCAATCCGTTGTTCAGGC-3′ ), SOCS6 (F: 5′ -AAGAATTCATCCCTTGGATTAGGTAAC-3′ , R: 5′ -CAGACTGGAGGTCGTGGAA-3′ ) 41 43 , and 3) absence of wheezing at auscultation, and, 4) first symptoms appearing within the last 14 days, and 5) radiological confirmation of pneumonia as per WHO guidelines 44 .", "Total mRNA was purified from transfected and infected MDMs using the RNeasy kit (Qiagen) and specific primers were used to amplify transforming growth factor beta-2 (TGFB2; F: 5′ -CCATCCCGCCCACTTTCTAC-3′ , R: 5′ -AGCTCAATCCGTTGTTCAGGC-3′ ), SOCS6 (F: 5′ -AAGAATTCATCCCTTGGATTAGGTAAC-3′ , R: 5′ -CAGACTGGAGGTCGTGGAA-3′ ) 41 43 , and 3) absence of wheezing at auscultation, and, 4) first symptoms appearing within the last 14 days, and 5) radiological confirmation of pneumonia as per WHO guidelines 44 . Based on these primary criteria defining pneumonia cases, all 74 cases were retrospectively re-evaluated according to the WHO \"Pocket book of hospital care for children\" 45 criteria to evaluate pneumonia severity. Cases that died during the study, or who had at least one additional clinical signs including central cyanosis, dullness to percussion during chest examination, prostration/lethargy, pleural effusion observed on chest radiography were retrospectively included in the severe pneumonia group.", "Cases that died during the study, or who had at least one additional clinical signs including central cyanosis, dullness to percussion during chest examination, prostration/lethargy, pleural effusion observed on chest radiography were retrospectively included in the severe pneumonia group. Patients without any of these additional clinical signs were included in the non-severe pneumonia group. Table 4 .", "Table 4 . a IP-10 values are expressed in pg ml -1 . IP-10 concentration differences between groups were compared using unpaired Mann-Whitney tests; significant changes (P < 0.05) are in bold. Clinical and molecular analysis. Nasopharyngeal aspirates (NAs) and whole blood samples were collected from children within 24 hours of admission.", "Nasopharyngeal aspirates (NAs) and whole blood samples were collected from children within 24 hours of admission. Whole blood samples were used for complete blood counts, blood culture and multiplex real-time PCR to identify Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae type B 46 . S. pneumoniae serotypes were defined using a 11 multiplex real-time PCR assay targeting the 40 most frequently represented serotypes or serogroups according to protocol developed by Messaoudi et al.", "S. pneumoniae serotypes were defined using a 11 multiplex real-time PCR assay targeting the 40 most frequently represented serotypes or serogroups according to protocol developed by Messaoudi et al. 47 . Serum C-reactive protein (CRP; AssayPro, St. Charles, Missouri, United States) and Procalcitonin (PCT; VIDAS B.R.A.H.M.S; bioMérieux) were quantified from whole-blood samples.", "Serum C-reactive protein (CRP; AssayPro, St. Charles, Missouri, United States) and Procalcitonin (PCT; VIDAS B.R.A.H.M.S; bioMérieux) were quantified from whole-blood samples. Multiplex real-time non quantitative PCR (Fast-Track Diagnostic, Sliema, Malta) was used to detect 19 viruses and five bacteria in the respiratory specimens (NAs and pleural effusions). Mixed detection was defined as 1) PCR-positive for multiple viruses in NAs, 2) positive blood culture or PCR-positive for multiple bacteria in blood or 3) PCR-positive for one or multiple viruses in NAs and one or multiple bacteria in blood (identified by PCR and blood culture).", "Mixed detection was defined as 1) PCR-positive for multiple viruses in NAs, 2) positive blood culture or PCR-positive for multiple bacteria in blood or 3) PCR-positive for one or multiple viruses in NAs and one or multiple bacteria in blood (identified by PCR and blood culture). Ethical approval. The study protocol, informed consent statement, clinical research form, any amendments and all other study documents were submitted to and approved by the Ethical Committee of the Instituto de Investigaciones en Ciencias de la Salud, the Universidad Nacional de Asunción (IICS-UNA) and the Hospital Pediátrico Niños de Acosta Ñu.", "The study protocol, informed consent statement, clinical research form, any amendments and all other study documents were submitted to and approved by the Ethical Committee of the Instituto de Investigaciones en Ciencias de la Salud, the Universidad Nacional de Asunción (IICS-UNA) and the Hospital Pediátrico Niños de Acosta Ñu. Informed consent was obtained from all subjects involved in this study. The clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki.", "The clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki. Statistical analysis. The Chi-square test and Fisher's exact test were used to compare categorical variables; continuous variables and non-normally distributed data were compared using the Mann-Whitney U-test; normally distributed data were compared using unpaired t-tests.", "The Chi-square test and Fisher's exact test were used to compare categorical variables; continuous variables and non-normally distributed data were compared using the Mann-Whitney U-test; normally distributed data were compared using unpaired t-tests. Comparative analyses between experimental conditions (i.e., MOCK, IAV, SP or IAV + SP) were performed using one-way ANOVA with Tukey's post-hoc test or Kruskal-Wallis analysis with Dunn's post-hoc tests. Receiver operating curve (ROC) analysis was used to determine the optimal cut-off value for IP-10 to differentiate between non-severe and severe pneumonia cases.", "Receiver operating curve (ROC) analysis was used to determine the optimal cut-off value for IP-10 to differentiate between non-severe and severe pneumonia cases. P < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism (La Jolla, California, United States)." ]

[ "Mixed viral and bacterial infections are widely described in community-acquired pneumonia; however, the clinical implications of co-infection on the associated immunopathology remain poorly studied. In this study, microRNA, mRNA and cytokine/chemokine secretion profiling were investigated for human monocyte-derived macrophages infected in-vitro with Influenza virus A/H1N1 and/or Streptococcus pneumoniae. We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions.", "We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions. We demonstrated that endogenous miRNA-200a-3p, whose expression was synergistically induced following co-infection, indirectly regulates CXCL10 expression by targeting suppressor of cytokine signaling-6 (SOCS-6), a well-known regulator of the JAK-STAT signaling pathway. Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5 years-old) hospitalized with viral and/or bacterial community-acquired pneumonia.", "Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5 years-old) hospitalized with viral and/or bacterial community-acquired pneumonia. Clinically, among the 74 cases of pneumonia, patients with identified mixed-detection had significantly higher (3.6-fold) serum IP-10 levels than those with a single detection (P = 0.03), and were significantly associated with severe pneumonia (P < 0.01). This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia.", "This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia. Text: Scientific RepoRts | 6:38532 | DOI: 10 .1038/srep38532 pathogenesis of several diseases and has been suggested as a potential biomarker of viral infection 10, 11 , late-onset bacterial infection in premature infants 12 , and a promising biomarker of sepsis and septic shock 13, 14 . Combined analysis of IP-10 and IFN-γ has also been reported as a useful biomarker for diagnosis and monitoring therapeutic efficacy in patients with active tuberculosis [15] [16] [17] , and both remain detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunction 18 .", "Combined analysis of IP-10 and IFN-γ has also been reported as a useful biomarker for diagnosis and monitoring therapeutic efficacy in patients with active tuberculosis [15] [16] [17] , and both remain detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunction 18 . With airway epithelial cells 19 , resident alveolar macrophages (AMs) and blood monocytes-derived macrophages (recruited into tissues under inflammatory conditions 20, 21 ) represent a major line of defense against both pneumococcal (through their high phagocytic capacity [22] [23] [24] ) and influenza infection 25, 26 . So far, no studies have yet focused on the intracellular mechanisms that regulate IP-10 in human blood leukocytes during mixed IAV and SP infection.", "So far, no studies have yet focused on the intracellular mechanisms that regulate IP-10 in human blood leukocytes during mixed IAV and SP infection. Several studies indicated that host non-coding small RNAs (including microRNAs) may function as immunomodulators by regulating several pivotal intracellular processes, such as the innate immune response 27 and antiviral activity 28, 29 ; both of these processes are closely related to toll-like receptor (TLR) signaling pathways. In this study, we firstly investigated the in vitro intracellular mechanisms that mediate the innate immune response in IAV and/or SP infected human monocyte-derived macrophages (MDMs).", "In this study, we firstly investigated the in vitro intracellular mechanisms that mediate the innate immune response in IAV and/or SP infected human monocyte-derived macrophages (MDMs). Using this approach, we observed that mixed-infection of MDMs induces a synergistic production of IP-10 which can be related to a miRNA-200a/JAK-STAT/SOCS-6 regulatory pathway. Subsequently, in a retrospective analysis of clinical samples collected from children ≤ 5 years-old hospitalized with pneumonia, we confirmed that serum IP-10 level could be related to both viral and/or bacterial etiologies and disease severity.", "Subsequently, in a retrospective analysis of clinical samples collected from children ≤ 5 years-old hospitalized with pneumonia, we confirmed that serum IP-10 level could be related to both viral and/or bacterial etiologies and disease severity. Characteristics of MDMs infected by IAV and/or SP. Initially, we investigated in vitro the impact of single and mixed IAV and SP infection on MDMs.", "Initially, we investigated in vitro the impact of single and mixed IAV and SP infection on MDMs. Firstly, active replication of IAV was assessed by qRT-PCR and quantification of new infectious viral particles in the cell supernatants ( Fig. 1a,b ).", "1a,b ). IAV titer increased over time after single infection with IAV and correlated with increased production of negative-strand IAV RNA. Maximum viral replication was observed at 18-24 hours post-infection, after which time both RNA replication and the quantity of infectious particles decreased.", "Maximum viral replication was observed at 18-24 hours post-infection, after which time both RNA replication and the quantity of infectious particles decreased. In this in vitro model, subsequent challenge of IAV-infected MDMs with SP had no significant impact on the production of new infectious viral particles (Fig. 1b) .", "1b) . Together, these results indicate permissive and productive infection of MDMs by IAV. Secondly, we evaluated whether MDMs are permissive for both IAV and SP infection. The presence of pneumococci within IAV-and SP-infected primary MDMs was confirmed at 8 h post-infection (Fig.", "The presence of pneumococci within IAV-and SP-infected primary MDMs was confirmed at 8 h post-infection (Fig. 1c) , suggesting that MDMs are permissive for viral and bacterial co-infection in the early steps of infection. Importantly, confocal co-detection of mixed IAV and SP was only effective following 8 h post-infection due to the bactericidal impact of SP internalization within human macrophages (after 24 h, data not shown).", "Importantly, confocal co-detection of mixed IAV and SP was only effective following 8 h post-infection due to the bactericidal impact of SP internalization within human macrophages (after 24 h, data not shown). Thirdly, we evaluated the impact of single and mixed infection with IAV and SP on MDM viability. Mixed infection significantly decreased cell viability (65.2 ± 4.5% total cell death at 48 hours post-infection; P < 0.0001) compared to single SP and IAV infection (39.6 ± 1.7% and 17.4 ± 1.1% total cell death, respectively; Fig.", "Mixed infection significantly decreased cell viability (65.2 ± 4.5% total cell death at 48 hours post-infection; P < 0.0001) compared to single SP and IAV infection (39.6 ± 1.7% and 17.4 ± 1.1% total cell death, respectively; Fig. 1d ). Taken together, these results confirmed human MDMs are permissive to mixed viral and bacterial infection.", "Taken together, these results confirmed human MDMs are permissive to mixed viral and bacterial infection. mRNA, microRNA and protein expression profiling reveal an overall induction of the host innate immune response following IAV and/or SP infection of MDMs. To investigate the innate immune response orchestrated by IAV-and SP-infected human MDMs, we firstly evaluated the expression of 84 genes involved in the innate and adaptive immune responses (Table S1) ; the major differentially-expressed genes are summarized in Fig.", "To investigate the innate immune response orchestrated by IAV-and SP-infected human MDMs, we firstly evaluated the expression of 84 genes involved in the innate and adaptive immune responses (Table S1) ; the major differentially-expressed genes are summarized in Fig. 2a . Expression profiling indicated an overall induction of genes related to the JAK-STAT, NF-Κ β and TLR signaling pathways.", "Expression profiling indicated an overall induction of genes related to the JAK-STAT, NF-Κ β and TLR signaling pathways. Indeed, all interferon-stimulated genes (ISGs) screened, including CXCL10 (fold-change [FC] = 240.9), CCL-2 (FC = 34.2) and MX-1 (FC = 151.4) were upregulated following mixed infection compared to uninfected cells, most of which are closely related to STAT-1 (FC = 52.3), IRF-7 (FC = 6.8) and IFNB1 (FC = 5.2) also found upregulated in mixed infected cells. Secondly, we investigated the endogenous microRNA expression profiles of IAV-and SP-infected MDMs.", "Secondly, we investigated the endogenous microRNA expression profiles of IAV-and SP-infected MDMs. A selection of microRNAs that were found to be differentially-expressed under different infection conditions are shown in Fig. 2b and Table S2 .", "2b and Table S2 . MiRNA-200a-3p was overexpressed after both single IAV (FC = 6.9), single SP (FC = 3.7) and mixed IAV/SP infection (FC = 7.3), indicating this miRNA may play a role in the innate immune response to viral and bacterial co-infection. Similar miRNA-200a-3p dysregulation profiles were obtained following IAV and/or SP infections of human macrophages-like (THP-1 monocytes-derived macrophages) or primary MDMs (data not shown).", "Similar miRNA-200a-3p dysregulation profiles were obtained following IAV and/or SP infections of human macrophages-like (THP-1 monocytes-derived macrophages) or primary MDMs (data not shown). Thirdly, the secreted levels of various antiviral, pro-inflammatory and immunomodulatory cytokines/chemokines were assayed in IAV-and SP-infected-THP-1 and primary MDM cell supernatants. We observed a remarkable correlation between the mRNA and protein expression profiles of single or mixed infected MDMs especially regarding CXCL-10 and IP-10 expression.", "We observed a remarkable correlation between the mRNA and protein expression profiles of single or mixed infected MDMs especially regarding CXCL-10 and IP-10 expression. Indeed, the level of IP-10 was synergistically increased in the supernatant of IAV-infected THP-1 MDMs exposed to SP (mean: 30,589 ± 16,484 pg ml −1 ) compared to single IAV infection (1,439 ± 566.5 pg ml −1 ) and single SP infection (4,472 ± 2,001 pg ml −1 ; P≤ 0.05; Fig. 2c ) at 24 hours after infection.", "2c ) at 24 hours after infection. In those cells, IP-10 expression reduced over time (48 to 72 hours), coinciding with a significant higher proportion of necrotic and apoptotic cells (Fig. 1d) . The synergistic expression of IP-10 was similarly observed at 24 hours post-infection using primary MDMs (Fig. 2d) .", "2d) . Significantly increased secretion of the other tested cytokines and chemokines was not observed post-infection, even in mixed infected MDMs (Fig. S1 ). Interestingly, a significant production of IP-10 was also observed in supernatants of primary human airway epithelial cells (HAEC) mixed-infected by IAV and SP compared to the single infections (Fig.", "Interestingly, a significant production of IP-10 was also observed in supernatants of primary human airway epithelial cells (HAEC) mixed-infected by IAV and SP compared to the single infections (Fig. 2e) . Taken together, the mRNA and protein profiling results suggested that mixed viral and bacterial infection of MDMs induces a synergistic pro-inflammatory response related to the type-1 interferon and JAK-STAT signaling pathways, with IP-10 as signature of IAV/SP co-infection.", "Taken together, the mRNA and protein profiling results suggested that mixed viral and bacterial infection of MDMs induces a synergistic pro-inflammatory response related to the type-1 interferon and JAK-STAT signaling pathways, with IP-10 as signature of IAV/SP co-infection. Among all microRNAs screened, miR-200a-3p was the most Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 overexpressed in IAV/SP co-infection of human MDMs. In the remainder of this study, we decided to investigate the interconnection between miR-200a-3p expression and the innate immune response.", "In the remainder of this study, we decided to investigate the interconnection between miR-200a-3p expression and the innate immune response. Endogenous miRNA-200a-3p expression correlates with CXCL10 (IP-10) induction following mixed IAV and SP infection of human MDMs. Using a specific Taqman probe assay targeting miR-200a-3p, we confirmed a significant upregulation of miR-200a-3p following mixed IAV and SP infection of human MDMs (Fig.", "Using a specific Taqman probe assay targeting miR-200a-3p, we confirmed a significant upregulation of miR-200a-3p following mixed IAV and SP infection of human MDMs (Fig. 3a) . In this experiment, a more marked up-regulation of miR-200a-3p was observed following IAV+ SP compared to results obtained previously (Fig. 2b) .", "2b) . This discrepancy has been attributed to the use of two different approaches to quantify miR-200a-3p expression. The use of a target-specific stem-loop reverse transcription primer in Fig. 3a allows a better sensitivity of miR-200a-3p detection compared to the non-specific fluorescent dye used in Fig. 2b .", "2b . As the general trend was suggestive of a synergistic induction of miR-200a-3p in response to mixed infection (Fig. 3a) , we hypothesized microRNA-200a-3p may play a role in the regulation of CXCL10 (IP-10), which was also synergistically upregulated in mixed-infected MDMs ( Fig.", "3a) , we hypothesized microRNA-200a-3p may play a role in the regulation of CXCL10 (IP-10), which was also synergistically upregulated in mixed-infected MDMs ( Fig. 2c and d) and primary HAEC ( Statistical analyses were performed using two-way ANOVA with Tukey's post-hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 CXCL10 (Fig.", "Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 CXCL10 (Fig. 3d) . These results suggested miR-200a-3p indirectly regulates CXCL10 and led us to hypothesize that miR-200a-3p controls a potential repressor of the JAK-STAT signaling pathway. . At 18 h after transfection, the MDMs were singly or mixed infected as described previously.", "At 18 h after transfection, the MDMs were singly or mixed infected as described previously. At 8 h post-IAV and/or SP infection, total mRNA was extracted and amplified by PCR using specific primers for the indicated genes. Values represent median ± IQR (a, c) or mean ± SEM (d, e) of three biological replicates.", "Values represent median ± IQR (a, c) or mean ± SEM (d, e) of three biological replicates. Statistical analyses were performed using a Kruskal-Wallis test (non-parametric, one-way ANOVA with Dunn's post-hoc test) for data presented in (a, c). An ordinary two-way ANOVA (with Tukey's post-hoc multiple comparison test) was used for data presented in (d, e).", "An ordinary two-way ANOVA (with Tukey's post-hoc multiple comparison test) was used for data presented in (d, e). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. MiRNA-200a-3p indirectly regulates IP-10 expression by targeting SOCS6.", "MiRNA-200a-3p indirectly regulates IP-10 expression by targeting SOCS6. As shown in Fig. 2a , several JAK-STAT signaling pathway genes were deregulated in mixed IAV-and SP-infected human MDMs; therefore, we hypothesized that miR-200a-3p directly regulates a regulator of the JAK-STAT signaling pathway.", "2a , several JAK-STAT signaling pathway genes were deregulated in mixed IAV-and SP-infected human MDMs; therefore, we hypothesized that miR-200a-3p directly regulates a regulator of the JAK-STAT signaling pathway. Predictive target analysis indicated that the 3' UTR of suppressor of cytokine signaling-6 (SOCS6) may be targeted by miR-200a-3p (Fig. 3b) .", "3b) . SOCS proteins constitute a class of negative regulators of JAK-STAT signaling pathways that are induced by both cytokines and TLR signaling. MiRNA-200a-3p was not predicted to target any of the other six members of the SOCS gene family.", "MiRNA-200a-3p was not predicted to target any of the other six members of the SOCS gene family. Transfection of human MDMs with MIM-200a downregulated SOCS6 (FC = 0.57) while inhibition of miR-200a-3p (INH-200a) upregulated SOCS6 (FC = 1.55), confirming that miR-200a-3p effectively regulates the expression of SOCS6 (Fig. 3e) .", "3e) . Moreover, SOCS6 was synergistically downregulated in IAV-or IAV/SP-infected MDMs overexpressing miRNA-200a (Fig. 3e) , suggesting that both infection and miR-200a-3p negatively regulate the expression of SOCS6. Finally, western blotting confirmed that expression of SOCS-6 sharply reduced following infection, especially after mixed IAV and SP infection (Fig. 3f) .", "3f) . These results indicate miR-200a-3p is strongly induced in response to mixed viral and bacterial co-infection, which in turn leads to downregulation of the JAK-STAT regulator SOCS-6 at both the mRNA and protein levels and subsequent upregulation of IP-10. analyses demonstrated mixed IAV and SP infection of human MDMs and HAEC induced significant production of IP-10.", "analyses demonstrated mixed IAV and SP infection of human MDMs and HAEC induced significant production of IP-10. As blood leukocytes and respiratory tract epithelial cells actively contribute to inflammation during pneumonia, we hypothesized the level of IP-10 in serum of patient with pneumonia may be both indicative of mixed respiratory infection and disease severity. As part of a prospective, hospital-based, multicenter case-control study on the etiology of pneumonia among children under 5-years-old, a total of 74 patients (44 male, 30 female) were included in this pilot evaluation.", "As part of a prospective, hospital-based, multicenter case-control study on the etiology of pneumonia among children under 5-years-old, a total of 74 patients (44 male, 30 female) were included in this pilot evaluation. According to WHO guidelines, retrospective analysis indicated 44 (59.5%) children had clinical signs of non-severe pneumonia and 30 (40.5%) children had signs of severe pneumonia. The main patient characteristics at inclusion are shown in Table 1 .", "The main patient characteristics at inclusion are shown in Table 1 . Patients with severe pneumonia had significant more recorded episodes of dyspnea (P < 0.001), cyanosis (P = 0.03), lower chest indrawing (P < 0.001), dullness to percussion (P < 0.001) and lethargy (P < 0.001) during chest examination than patient with non-severe pneumonia. Moreover, pleural effusions were significantly more observed among critically ill patients and the duration of hospitalization was significantly longer for the children with severe pneumonia than for those with non-severe pneumonia (P = 0.0015).", "Moreover, pleural effusions were significantly more observed among critically ill patients and the duration of hospitalization was significantly longer for the children with severe pneumonia than for those with non-severe pneumonia (P = 0.0015). Two deaths occurred within the group of children retrospectively defined with severe pneumonia. Evaluation of the systemic inflammatory response of the 74 cases is shown in Table 2 .", "Evaluation of the systemic inflammatory response of the 74 cases is shown in Table 2 . Serum level of CRP, IP-10, PCT, G-CSF, IL-6, IL-8 and MIP-1β were significantly more elevated in serum samples from critically ill patients. Patients with severe pneumonia had significantly higher (4.2-fold) serum IP-10 levels than those with a non-severe pneumonia (P < 0.001) suggesting IP-10 as a promising prognostic marker in pneumonia.", "Patients with severe pneumonia had significantly higher (4.2-fold) serum IP-10 levels than those with a non-severe pneumonia (P < 0.001) suggesting IP-10 as a promising prognostic marker in pneumonia. Diagnostic accuracy measures for predicting pneumonia severity using blood-based biomarkers are summarized in Table S3 . Briefly, in this study, the optimal IP-10 cut-off value for identifying patient with severe pneumonia was 4,240 pg ml −1 , with an area under the receiver operating characteristic curve of 0.69 (95% CI, 0.57 to 0.82, P < 0.001).", "Briefly, in this study, the optimal IP-10 cut-off value for identifying patient with severe pneumonia was 4,240 pg ml −1 , with an area under the receiver operating characteristic curve of 0.69 (95% CI, 0.57 to 0.82, P < 0.001). Defining as positive a serum IP-10 level above this cut-off resulted in a sensitivity of 63.3%, specificity of 63.6% and a positive likelihood ratio of 1.74. Prognostic values of IP-10 were closed to procalcitonin (PCT; AUC = 0.70; 95% IC, 0.58 to 0.82, P < 0.001) and IL-6 (AUC = 0.70; 95% IC, 0.58-0.83, P < 0.001).", "Prognostic values of IP-10 were closed to procalcitonin (PCT; AUC = 0.70; 95% IC, 0.58 to 0.82, P < 0.001) and IL-6 (AUC = 0.70; 95% IC, 0.58-0.83, P < 0.001). Multiplex PCR-based screening of respiratory and blood samples reveal a high variety of pathogen associations (Table 3) . Respiratory viruses were detected in the nasal aspirates (NAs) of 63/74 patients (85.1%).", "Respiratory viruses were detected in the nasal aspirates (NAs) of 63/74 patients (85.1%). Etiological bacteria of pneumonia (S. pneumoniae, n = 19; S. aureus, n = 1; or H. influenzae type B, n = 7) were identified via real-time PCR in the blood samples of 27/74 (36.5%) of the patients. Multiplex PCR assays allowed the identification of respiratory bacteria in the blood of 19 patients with negative blood culture results.", "Multiplex PCR assays allowed the identification of respiratory bacteria in the blood of 19 patients with negative blood culture results. Among the 74 cases PCR-positive for respiratory pathogens, a single virus or bacteria were detected in the NAs of 7 (9.4%) and 3 (4.0%) patients, respectively; these 10/74 (13.5%) cases were defined as the single infection group. The mixed infection group included the 62/74 (83.8%) cases in which (1) multiple viruses and/or bacteria were identified in NAs (38/74; 51.3%) without any bacteria identified in blood samples or (2) one or more viruses and/or bacteria were identified in NAs and associated with a blood bacteremia (24/74; 32.4%).", "The mixed infection group included the 62/74 (83.8%) cases in which (1) multiple viruses and/or bacteria were identified in NAs (38/74; 51.3%) without any bacteria identified in blood samples or (2) one or more viruses and/or bacteria were identified in NAs and associated with a blood bacteremia (24/74; 32.4%). We evaluated whether IP-10 serum level could correlate with the viral and bacterial etiologies of pneumonia. Patients with mixed infection had significant higher (3.6-fold) IP-10 serum level than patient with single detection (P = 0.03; Table 4 ).", "Patients with mixed infection had significant higher (3.6-fold) IP-10 serum level than patient with single detection (P = 0.03; Table 4 ). A stratified analysis reveals that the highest IP-10 serum level was observed among patients with both several respiratory pathogens identified (mixed-detection group) and severe pneumonia (14,427 pg ml −1 , IQR (3,981-82,994). In detail, a remarkable IP-10 serum level (142,531 pg ml −1 ), representing 33-fold higher above cut-off value predicting pneumonia severity was observed in patient with hRV in NA co-detected with S. pneumoniae (serotype 14) in pleural effusion and blood.", "In detail, a remarkable IP-10 serum level (142,531 pg ml −1 ), representing 33-fold higher above cut-off value predicting pneumonia severity was observed in patient with hRV in NA co-detected with S. pneumoniae (serotype 14) in pleural effusion and blood. In concordance with our in-vitro model of co-infection, a significant IP-10 level (90,338 pg ml −1 ) was quantified in blood sample of patient with severe bacteremic pneumococcal (serotype 14) pneumonia with a positive co-detection of Influenza B virus in NA. Taken together, these results suggest that high serum IP-10 levels are significantly associated with mixed viral and bacterial detection and also related to pneumonia pathogenesis.", "Taken together, these results suggest that high serum IP-10 levels are significantly associated with mixed viral and bacterial detection and also related to pneumonia pathogenesis. This study provides additional in vitro and clinical data to improve our understanding of the immunopathology of mixed viral and bacterial pneumonia (Fig. 4) .", "4) . The in vitro model of influenza and pneumococcal superinfection of human MDMs demonstrated that mixed infection synergistically induced release of the pro-inflammatory chemokine IP-10, strongly suggesting human Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 blood leukocytes contribute to the immunopathology of pneumonia. Additionally, transcriptomics and omics analyses provided new data on the inflammatory pathways that are activated during mixed infection and related to synergistic induction of the pro-inflammatory chemokine IP-10 in mixed infected cells.", "Additionally, transcriptomics and omics analyses provided new data on the inflammatory pathways that are activated during mixed infection and related to synergistic induction of the pro-inflammatory chemokine IP-10 in mixed infected cells. Our observations are consistent with a recent study describing IP-10 induction as host-proteome signature of both viral and bacterial infections 30 . Of the differentially-expressed genes observed in mixed infected MDMs, the transcription factors STAT-1 and IRF-7 appear to play crucial roles in the regulation of interferon-stimulated genes including CXCL10 (IP-10).", "Of the differentially-expressed genes observed in mixed infected MDMs, the transcription factors STAT-1 and IRF-7 appear to play crucial roles in the regulation of interferon-stimulated genes including CXCL10 (IP-10). By focusing on the intracellular mechanisms that regulate inflammatory pathways, we demonstrated a novel role for miRNA-200a-3p in the regulation of CXCL10 (IP-10). These observations are consistent with previous reports showing that RNA virus infection upregulates miR-155 in macrophages and dendritic cells and also regulates suppressor of cytokine signaling 1 (SOCS1), suggesting the existence of a miRNA/JAK-STAT/SOCS regulatory pathway during viral infection 29 .", "These observations are consistent with previous reports showing that RNA virus infection upregulates miR-155 in macrophages and dendritic cells and also regulates suppressor of cytokine signaling 1 (SOCS1), suggesting the existence of a miRNA/JAK-STAT/SOCS regulatory pathway during viral infection 29 . Our study suggests co-infection leads to overexpression of miR-200a-3p, which in turn targets and downregulates the JAK-STAT regulator SOCS-6 and consequently increases CXCL10 (IP-10) expression. Interestingly, a complementary in-silico approach reveals that several microRNAs that were found dysregulated in our experiments of IAV and SP co-infection of MDMs or HAEC, might target several genes of SOCS family and play similar role than miR-200a-3p.", "Interestingly, a complementary in-silico approach reveals that several microRNAs that were found dysregulated in our experiments of IAV and SP co-infection of MDMs or HAEC, might target several genes of SOCS family and play similar role than miR-200a-3p. Indeed, miRNA-142-3p might target SOCS4, 5, 6 mRNA while miRNA-194-5p might target SOCS2, 3, 4, 5 and 7 mRNA. These observations underline that intra-cellular regulation of IP-10 is not limited to the contribution of a sole microRNA.", "These observations underline that intra-cellular regulation of IP-10 is not limited to the contribution of a sole microRNA. A complex inter-relationship between numerous host microRNAs and inhibitors of the JAK-STAT signaling pathway occur to control host innate inflammatory response against viral and/or bacterial infections. Clinically, the majority of pediatric CAP cases in this study were associated with both positive viral and/or bacterial detection.", "Clinically, the majority of pediatric CAP cases in this study were associated with both positive viral and/or bacterial detection. Respiratory microorganisms were detected in 97% of cases; 51.3% of which were viral-viral, viral-bacterial or bacterial-bacterial co-detected only in nasal aspirates, 32.4% of which co-detected in both nasal aspirates and blood samples. These data are consistent with previous etiological studies of pediatric CAP 3,31-33 .", "These data are consistent with previous etiological studies of pediatric CAP 3,31-33 . S. pneumoniae was the major bacteria identified in blood (19/74; 25.7%) and mainly co-detected with respiratory viruses in NAs (16/19; 84.2%). We observed a very high diversity of viral and bacterial associations in biological samples from children with pneumonia.", "We observed a very high diversity of viral and bacterial associations in biological samples from children with pneumonia. In comparison with IAV and SP14 combination evaluated in-vitro, no pneumonia cases were singly influenza and pneumococcus infected, and no similar co-detection with those two pathogens has been clinically observed. Nevertheless, Influenza B (IVB) virus was identified in 5 patients and two of them had a positive SP co-detection in blood (one non-typable strain and one serotype 14 using our molecular typing test).", "Nevertheless, Influenza B (IVB) virus was identified in 5 patients and two of them had a positive SP co-detection in blood (one non-typable strain and one serotype 14 using our molecular typing test). IVB and SP14 combination seems to be the nearest pathogen co-detection to that in-vitro investigated. Clinically, this co-detection was associated with both a very high IP-10 expression and a very severe pneumonia case definition.", "Clinically, this co-detection was associated with both a very high IP-10 expression and a very severe pneumonia case definition. Interestingly, our translational pilot evaluation reveals IP-10 expression can be induced by several different viral and/or bacterial combinations. As immune response to each pathogen is different, further in-vitro investigations using different pathogens associations are needed to better characterize the mechanisms involved in the immunopathology of pneumonia.", "As immune response to each pathogen is different, further in-vitro investigations using different pathogens associations are needed to better characterize the mechanisms involved in the immunopathology of pneumonia. In this cohort, highest serum IP-10 levels were identified among patients with both several pathogen detected and severe pneumonia, suggesting a significant role of IP-10 on pneumonia pathogenesis. Indeed, high plasma levels of IP-10 have previously been reported in patients with sepsis 12 , and were associated with high mortality rate, especially among patients with CAP 34 .", "Indeed, high plasma levels of IP-10 have previously been reported in patients with sepsis 12 , and were associated with high mortality rate, especially among patients with CAP 34 . Additionally, the IP-10-CXCR3 axis has been related to acute immune lung injury and lymphocyte apoptosis during the development of severe acute respiratory syndrome (SARS) 35, 36 . Moreover, an in vivo study that modeled influenza and pneumococcal superinfection in mice indicated that pro-inflammatory chemokines, including IP-10, play a crucial role in influenza-induced susceptibility to lung neutrophilia, severe immunopathology and mortality 37 .", "Moreover, an in vivo study that modeled influenza and pneumococcal superinfection in mice indicated that pro-inflammatory chemokines, including IP-10, play a crucial role in influenza-induced susceptibility to lung neutrophilia, severe immunopathology and mortality 37 . In this study, markedly elevated IP-10 (92,809 pg ml −1 ) combined with the highest PCT level (74.4 pg ml −1 ) were quantified in the serum sample of a child who died, in whom S. pneumoniae (serotype 9 V) was identified in the blood (PCR and blood culture) and co-detected with Haemophilus influenzae type B in nasal aspirate. These observations suggest an interrelationship between co-detection, elevated serum IP-10 and the pathogenesis of pneumonia.", "These observations suggest an interrelationship between co-detection, elevated serum IP-10 and the pathogenesis of pneumonia. Several limitations of this pilot translational study need to be acknowledged before concluding mixed infection is related to elevated IP-10 and disease severity. Indeed, although viral shedding (e.g., of HRV and HBoV) is common in asymptomatic children, we were unable to evaluate the levels of immunomodulators in the serum samples of a control group.", "Indeed, although viral shedding (e.g., of HRV and HBoV) is common in asymptomatic children, we were unable to evaluate the levels of immunomodulators in the serum samples of a control group. Moreover, although the samples were collected within the first 24 hours after admission, only a single blood sample was processed for each patient. Therefore, a larger, longitudinal study on the etiology and severity of pneumonia will be necessary to confirm these results.", "Therefore, a larger, longitudinal study on the etiology and severity of pneumonia will be necessary to confirm these results. In conclusion, the present findings suggest that mixed respiratory infections and IP-10 may play major, interconnected roles in the pathogenesis of pneumonia. Clinically, assessment and monitoring of induced IP-10 serum level may assist clinicians to improve diagnosis and patient management of severe community-acquired pneumonia.", "Clinically, assessment and monitoring of induced IP-10 serum level may assist clinicians to improve diagnosis and patient management of severe community-acquired pneumonia. Viral and bacterial strains. The 10 ng ml −1 M-CSF (Miltenyi Biotec).", "Viral and bacterial strains. The 10 ng ml −1 M-CSF (Miltenyi Biotec). THP− 1 MDMs were obtained by culturing cells with 10 ng ml -1 phorbol myristate acetate (PMA; Invivogen, Toulouse, France) for 72 hours.", "THP− 1 MDMs were obtained by culturing cells with 10 ng ml -1 phorbol myristate acetate (PMA; Invivogen, Toulouse, France) for 72 hours. Human airway epithelial cells (HAEC, bronchial cell type) originated from a 54-years old woman with no pathology reported (batch number MD056501) were provided by Mucilair (Epithelix, Geneva, Switzerland). Sterility, tissue integrity (TEER), mucus production and cilia beating frequency have been certified by the company.", "Sterility, tissue integrity (TEER), mucus production and cilia beating frequency have been certified by the company. Gene expression profiling. Total cellular mRNA was purified using the RNeasy kit (Qiagen, Hilden, Germany). Reverse-transcription of total mRNA was performed using the RT 2 First Strand Kit (SABiosciences, Hilden, Germany).", "Reverse-transcription of total mRNA was performed using the RT 2 First Strand Kit (SABiosciences, Hilden, Germany). The expression of 84 genes involved in the human innate and adaptive immune responses was evaluated using the RT 2 profiler ™ PCR Array (SABiosciences) according to the manufacturer's recommendations. The Δ Δ Ct method was applied to calculate the fold changes in gene expression for each gene relative to uninfected control cells using the web-based RT 2 profiler PCR Array Data Analysis software (SABiosciences).", "The Δ Δ Ct method was applied to calculate the fold changes in gene expression for each gene relative to uninfected control cells using the web-based RT 2 profiler PCR Array Data Analysis software (SABiosciences). MicroRNA profiling array. Total cellular microRNAs were purified using the miRNeasy Mini kit (Qiagen) and reverse-transcribed using the miScript Reverse Transcription kit (Qiagen).", "Total cellular microRNAs were purified using the miRNeasy Mini kit (Qiagen) and reverse-transcribed using the miScript Reverse Transcription kit (Qiagen). The profiling of 84 miRNAs was performed using the Human Immunopathology miScript miRNA PCR Array kit (Qiagen) according to the manufacturer's instructions. Data were analyzed using the miScript miRNA PCR array data analysis web portal.", "Data were analyzed using the miScript miRNA PCR array data analysis web portal. In silico miRNA target prediction. MiRNA target genes were retrieved and compiled using TargetScan 38 and microRNA.org resource 39 . The interactions between miRNAs and intracellular pathways were predicted using DIANA-miRPath v2.0 40 .", "The interactions between miRNAs and intracellular pathways were predicted using DIANA-miRPath v2.0 40 . THP-1 MDMs were seeded in 24-well plates (0.5 × 10 6 per well) in triplicate, exposed to Influenza A H1N1 (A/Solomon islands/3/2006) virus (IAV) under serum-free conditions for 1 hour and then cultured for 3 hours in fresh RPMI-1640 containing 2% FBS. Streptococcus pneumoniae (SP) serotype 14 was added at 4 hours after IAV infection.", "Streptococcus pneumoniae (SP) serotype 14 was added at 4 hours after IAV infection. Gentamicin (10 μ g ml −1 ) was added 2 hours after SP infection (i.e. 6 hours post-influenza infection) and maintained in the culture media throughout the experiment to kill extracellular bacteria and limit bacterial growth.", "6 hours post-influenza infection) and maintained in the culture media throughout the experiment to kill extracellular bacteria and limit bacterial growth. Cell viability was determined by flow-cytometry using the FITC/Annexin V apoptosis detection kit (BD Biosciences), according to the manufacturer's instructions. #4427975) .", "#4427975) . In this assay, fold changes have been defined by the Δ Δ Ct method using control RNU-44 and -48 as reference microRNAs. Total mRNA was purified from transfected and infected MDMs using the RNeasy kit (Qiagen) and specific primers were used to amplify transforming growth factor beta-2 (TGFB2; F: 5′ -CCATCCCGCCCACTTTCTAC-3′ , R: 5′ -AGCTCAATCCGTTGTTCAGGC-3′ ), SOCS6 (F: 5′ -AAGAATTCATCCCTTGGATTAGGTAAC-3′ , R: 5′ -CAGACTGGAGGTCGTGGAA-3′ ) 41 43 , and 3) absence of wheezing at auscultation, and, 4) first symptoms appearing within the last 14 days, and 5) radiological confirmation of pneumonia as per WHO guidelines 44 .", "Total mRNA was purified from transfected and infected MDMs using the RNeasy kit (Qiagen) and specific primers were used to amplify transforming growth factor beta-2 (TGFB2; F: 5′ -CCATCCCGCCCACTTTCTAC-3′ , R: 5′ -AGCTCAATCCGTTGTTCAGGC-3′ ), SOCS6 (F: 5′ -AAGAATTCATCCCTTGGATTAGGTAAC-3′ , R: 5′ -CAGACTGGAGGTCGTGGAA-3′ ) 41 43 , and 3) absence of wheezing at auscultation, and, 4) first symptoms appearing within the last 14 days, and 5) radiological confirmation of pneumonia as per WHO guidelines 44 . Based on these primary criteria defining pneumonia cases, all 74 cases were retrospectively re-evaluated according to the WHO \"Pocket book of hospital care for children\" 45 criteria to evaluate pneumonia severity. Cases that died during the study, or who had at least one additional clinical signs including central cyanosis, dullness to percussion during chest examination, prostration/lethargy, pleural effusion observed on chest radiography were retrospectively included in the severe pneumonia group.", "Cases that died during the study, or who had at least one additional clinical signs including central cyanosis, dullness to percussion during chest examination, prostration/lethargy, pleural effusion observed on chest radiography were retrospectively included in the severe pneumonia group. Patients without any of these additional clinical signs were included in the non-severe pneumonia group. Table 4 .", "Table 4 . a IP-10 values are expressed in pg ml -1 . IP-10 concentration differences between groups were compared using unpaired Mann-Whitney tests; significant changes (P < 0.05) are in bold. Clinical and molecular analysis. Nasopharyngeal aspirates (NAs) and whole blood samples were collected from children within 24 hours of admission.", "Nasopharyngeal aspirates (NAs) and whole blood samples were collected from children within 24 hours of admission. Whole blood samples were used for complete blood counts, blood culture and multiplex real-time PCR to identify Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae type B 46 . S. pneumoniae serotypes were defined using a 11 multiplex real-time PCR assay targeting the 40 most frequently represented serotypes or serogroups according to protocol developed by Messaoudi et al.", "S. pneumoniae serotypes were defined using a 11 multiplex real-time PCR assay targeting the 40 most frequently represented serotypes or serogroups according to protocol developed by Messaoudi et al. 47 . Serum C-reactive protein (CRP; AssayPro, St. Charles, Missouri, United States) and Procalcitonin (PCT; VIDAS B.R.A.H.M.S; bioMérieux) were quantified from whole-blood samples.", "Serum C-reactive protein (CRP; AssayPro, St. Charles, Missouri, United States) and Procalcitonin (PCT; VIDAS B.R.A.H.M.S; bioMérieux) were quantified from whole-blood samples. Multiplex real-time non quantitative PCR (Fast-Track Diagnostic, Sliema, Malta) was used to detect 19 viruses and five bacteria in the respiratory specimens (NAs and pleural effusions). Mixed detection was defined as 1) PCR-positive for multiple viruses in NAs, 2) positive blood culture or PCR-positive for multiple bacteria in blood or 3) PCR-positive for one or multiple viruses in NAs and one or multiple bacteria in blood (identified by PCR and blood culture).", "Mixed detection was defined as 1) PCR-positive for multiple viruses in NAs, 2) positive blood culture or PCR-positive for multiple bacteria in blood or 3) PCR-positive for one or multiple viruses in NAs and one or multiple bacteria in blood (identified by PCR and blood culture). Ethical approval. The study protocol, informed consent statement, clinical research form, any amendments and all other study documents were submitted to and approved by the Ethical Committee of the Instituto de Investigaciones en Ciencias de la Salud, the Universidad Nacional de Asunción (IICS-UNA) and the Hospital Pediátrico Niños de Acosta Ñu.", "The study protocol, informed consent statement, clinical research form, any amendments and all other study documents were submitted to and approved by the Ethical Committee of the Instituto de Investigaciones en Ciencias de la Salud, the Universidad Nacional de Asunción (IICS-UNA) and the Hospital Pediátrico Niños de Acosta Ñu. Informed consent was obtained from all subjects involved in this study. The clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki.", "The clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki. Statistical analysis. The Chi-square test and Fisher's exact test were used to compare categorical variables; continuous variables and non-normally distributed data were compared using the Mann-Whitney U-test; normally distributed data were compared using unpaired t-tests.", "The Chi-square test and Fisher's exact test were used to compare categorical variables; continuous variables and non-normally distributed data were compared using the Mann-Whitney U-test; normally distributed data were compared using unpaired t-tests. Comparative analyses between experimental conditions (i.e., MOCK, IAV, SP or IAV + SP) were performed using one-way ANOVA with Tukey's post-hoc test or Kruskal-Wallis analysis with Dunn's post-hoc tests. Receiver operating curve (ROC) analysis was used to determine the optimal cut-off value for IP-10 to differentiate between non-severe and severe pneumonia cases.", "Receiver operating curve (ROC) analysis was used to determine the optimal cut-off value for IP-10 to differentiate between non-severe and severe pneumonia cases. P < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism (La Jolla, California, United States)." ]

[ "The common marmoset (Callithrix jacchus) is increasingly being utilised as a nonhuman primate model for human disease, ranging from autoimmune to infectious disease. In order to fully exploit these models, meaningful comparison to the human host response is necessary. Commercially available reagents, primarily targeted to human cells, were utilised to assess the phenotype and activation status of key immune cell types and cytokines in naive and infected animals.", "Commercially available reagents, primarily targeted to human cells, were utilised to assess the phenotype and activation status of key immune cell types and cytokines in naive and infected animals. Single cell suspensions of blood, spleen, and lung were examined. Generally, the phenotype of cells was comparable between humans and marmosets, with approximately 63% of all lymphocytes in the blood of marmosets being T cells, 25% B-cells, and 12% NK cells.", "Generally, the phenotype of cells was comparable between humans and marmosets, with approximately 63% of all lymphocytes in the blood of marmosets being T cells, 25% B-cells, and 12% NK cells. The percentage of neutrophils in marmoset blood were more similar to human values than mouse values. Comparison of the activation status of cells following experimental systemic or inhalational infection exhibited different trends in different tissues, most obvious in cell types active in the innate immune response.", "Comparison of the activation status of cells following experimental systemic or inhalational infection exhibited different trends in different tissues, most obvious in cell types active in the innate immune response. This work significantly enhances the ability to understand the immune response in these animals and fortifies their use as models of infectious disease. Text: The common marmoset (Callithrix jacchus), a New World monkey (NWM) species is a small, arboreal nonhuman primate (NHP), native to the Atlantic Coastal Forest in Northeast Brazil and parts of South East Brazil.", "Text: The common marmoset (Callithrix jacchus), a New World monkey (NWM) species is a small, arboreal nonhuman primate (NHP), native to the Atlantic Coastal Forest in Northeast Brazil and parts of South East Brazil. In recent years the common marmoset has become more widely used in applied biomedical research, and an increasing body of evidence suggests the physiological and immunological responses to biological insults are similar between marmosets and humans [1] . In the field of infectious disease, the marmoset is primarily being investigated as an alternative NHP model to complement the more traditionally used Old World monkeys (OWM) (e.g., rhesus and cynomolgus macaques).", "In the field of infectious disease, the marmoset is primarily being investigated as an alternative NHP model to complement the more traditionally used Old World monkeys (OWM) (e.g., rhesus and cynomolgus macaques). Evolutionarily, both NWM and OWM sit within the simiiformes infraorder of the suborder Haplorhini of primates [2] . Marmosets sit within the family Callitrichidae of the Platyrrhini parvorder, while OWM sit within the Cercopithecidae family of the Catarrhini Parvorder.", "Marmosets sit within the family Callitrichidae of the Platyrrhini parvorder, while OWM sit within the Cercopithecidae family of the Catarrhini Parvorder. Marmosets therefore are separated from Old World monkeys by one ancestral step and are a lower order primate. Marmosets have been used to model the infection syndrome caused by a number of public health pathogens including Lassa virus [3] , Hepatitis C virus [4] , Dengue virus [5] , Herpesvirus [6] , Junin virus [7] Rift Valley Fever [8] , and SARS [9] .", "Marmosets have been used to model the infection syndrome caused by a number of public health pathogens including Lassa virus [3] , Hepatitis C virus [4] , Dengue virus [5] , Herpesvirus [6] , Junin virus [7] Rift Valley Fever [8] , and SARS [9] . Marmosets have also been used to model a number of biodefense pathogens including Eastern Equine Encephalitis virus [10] , Bacillus anthracis [11] , Francisella tularensis [12, 13] , Burkholderia pseudomallei [14] , Marburg haemorrhagic fever virus [15, 16] , Ebola haemorrhagic fever virus [16] , and Variola virus [17] . The utility of marmosets to assess medical countermeasures has also been demonstrated; a vaccine has been tested for Lassa fever [18] and the efficacy of ciprofloxacin and levofloxacin has been tested as postexposure therapies for anthrax and tularemia, respectively [19, 20] .", "The utility of marmosets to assess medical countermeasures has also been demonstrated; a vaccine has been tested for Lassa fever [18] and the efficacy of ciprofloxacin and levofloxacin has been tested as postexposure therapies for anthrax and tularemia, respectively [19, 20] . In order to exploit these models fully and to allow meaningful comparison with the human condition, the response of the immune system to infection/therapy needs to be 2 Journal of Immunology Research characterised and understood. Generally, NHPs have a close molecular, immunological, reproductive, and neurological similarity with humans making them ideal surrogates for humans and the study of infectious diseases.", "Generally, NHPs have a close molecular, immunological, reproductive, and neurological similarity with humans making them ideal surrogates for humans and the study of infectious diseases. There is a high level of gene homology between humans and NHPs which underlies physiological and biochemical similarities. Similarities at the genetic level extend to the phenotypical level making NHPs well suited to modelling pathophysiological responses in man [21] .", "Similarities at the genetic level extend to the phenotypical level making NHPs well suited to modelling pathophysiological responses in man [21] . Immunologically, there is a high degree of homology between humans and marmosets [22] . The similarity of various immunological factors produced by humans and marmosets has been investigated at both the genetic and protein levels.", "The similarity of various immunological factors produced by humans and marmosets has been investigated at both the genetic and protein levels. There is at least 95% homology between human costimulatory molecules (e.g., CD80, CD86 etc.) and those of marmosets [23] .", "and those of marmosets [23] . Also the immunoglobulin and T-cell receptor repertoire of humans and marmosets show at least 80% homology [24, 25] . Currently, the availability of commercial reagents specifically designed for the marmoset is limited although a number of antibodies designed for use with human samples have been shown to cross-react with leucocytes from marmoset blood [26] [27] [28] .", "Currently, the availability of commercial reagents specifically designed for the marmoset is limited although a number of antibodies designed for use with human samples have been shown to cross-react with leucocytes from marmoset blood [26] [27] [28] . However, these reagents have not been exploited to investigate the immune response to infectious disease. To date, investigation of the immune response in marmosets has primarily been achieved using pathogen-specific antibodies to determine the serological response using ELISA such as in the smallpox, Dengue, Rift Valley Fever, and Herpes models [5, 6, 8, 17] or by immunohistochemistry to identify, for example, CD8+, CD3+, CD20+ cells, and IL-6 in the smallpox model [17] ; neutrophils and macrophages in the Herpes model [6] ; or CD3+ and CD20+ cells in the Lassa model [3] .", "To date, investigation of the immune response in marmosets has primarily been achieved using pathogen-specific antibodies to determine the serological response using ELISA such as in the smallpox, Dengue, Rift Valley Fever, and Herpes models [5, 6, 8, 17] or by immunohistochemistry to identify, for example, CD8+, CD3+, CD20+ cells, and IL-6 in the smallpox model [17] ; neutrophils and macrophages in the Herpes model [6] ; or CD3+ and CD20+ cells in the Lassa model [3] . The work presented here focuses on understanding the immune profile of the naive marmoset as well as identifying and quantifying the immune response to infectious disease. The aim of this work is to determine key changes and identify correlates of infection or protection.", "The aim of this work is to determine key changes and identify correlates of infection or protection. Healthy sexually mature common marmosets (C. jacchus) were obtained from the Dstl Porton Down breeding colony and housed in vasectomized male and female pairs. The Dstl colony was established during the 1970s and is a closed colony with a stable genotype.", "The Dstl colony was established during the 1970s and is a closed colony with a stable genotype. Animals included in these studies were mixed sex pairs, between 18 months and 5 years old and weighing between 320 g to 500 g. All animals were allowed free access to food and water as well as environmental enrichment. All animal studies were carried out in accordance with the UK Animals (Scientific Procedures) Act of 1986 and the Codes of Practice for the Housing and Care of Animals used in Scientific Procedures 1989.", "All animal studies were carried out in accordance with the UK Animals (Scientific Procedures) Act of 1986 and the Codes of Practice for the Housing and Care of Animals used in Scientific Procedures 1989. Animals were challenged with an intracellular pathogen by either the subcutaneous or inhalational route and were humanely killed at various time points after challenge. Prior to the infection study, animals were bled to determine baseline immunological parameters.", "Prior to the infection study, animals were bled to determine baseline immunological parameters. Studies were performed to establish infection models in order to evaluate the efficacy of suitable therapies for transition ultimately to the clinic. Populations. Blood and tissue samples were homogenised to provide single cell suspensions [12] .", "Blood and tissue samples were homogenised to provide single cell suspensions [12] . Red blood cells were lysed, and the mixed leucocyte population was washed and stained with various combinations of the following fluorescent antibody stains: CD3 (SP34-2), CD8 (LT8), CD11c (SHCL3), CD14 (M5E2), CD16 (3G8), CD20 (Bly1), CD45RA (5H9), CD54 (HCD54), CD56 (B159), CD69 (FN50), CD163 (GHI/61), and MCHII (L243) (BD Bioscience, Insight Bioscience, AbD serotec). Samples were fixed in 4% paraformaldehyde for 48 hrs at 4 ∘ C and analysed by flow cytometry (FACScanto II BD) within 72 hours of staining.", "Samples were fixed in 4% paraformaldehyde for 48 hrs at 4 ∘ C and analysed by flow cytometry (FACScanto II BD) within 72 hours of staining. Levels of circulating cytokines and chemokines were also quantified in the blood of marmosets from the Dstl colony using human multiplex kits available commercially (BD cytokine flex beads and the Luminex system). These systems show significant cross-reactivity with the marmoset suggesting a high degree of conservation between the two species for IL-6, MIP-1 , MIP-1 , and MCP-1 [29] .", "These systems show significant cross-reactivity with the marmoset suggesting a high degree of conservation between the two species for IL-6, MIP-1 , MIP-1 , and MCP-1 [29] . However, for other cytokines that are pivotal in the innate response, TNF and IFN reagents were obtained from U-CyTech Biosciences and Mabtech AB, respectively, due to a lack of cross-reactivity observed within the kit obtained from BD [13] . In order to fully characterise the immune response to infectious agent in the marmoset, single cell suspensions of lung and spleen tissue were also examined in conjunction with the traditionally used blood cells.", "In order to fully characterise the immune response to infectious agent in the marmoset, single cell suspensions of lung and spleen tissue were also examined in conjunction with the traditionally used blood cells. These tissue homogenates are of particular interest in relation to target sites of infection: the lung as the site of initial infection following an inhalational challenge and the spleen as a representative organ following a parental challenge. Cell types targeted during this analysis include cells important in the innate response (e.g., neutrophils, macrophages, and NK cells) and the adaptive response (T and B cells) with a view to determine the response to infection and vaccination and to derive immune correlates of infection/protection.", "Cell types targeted during this analysis include cells important in the innate response (e.g., neutrophils, macrophages, and NK cells) and the adaptive response (T and B cells) with a view to determine the response to infection and vaccination and to derive immune correlates of infection/protection. Dapi was included as a nuclear marker to ensure that the initial gating included only intact cells. Basic cell types in blood were easily identified by measuring size (forward) and granularity (side) scatter (Figure 1(a) ).", "Basic cell types in blood were easily identified by measuring size (forward) and granularity (side) scatter (Figure 1(a) ). Identification of cell types in tissue samples was more difficult as the scatter profiles are less clearly compartmentalized. The common leukocyte antigen (CD45) normally used to locate all leukocytes in human samples also worked well in marmoset blood but failed to provide relevant information in the tissue samples.", "The common leukocyte antigen (CD45) normally used to locate all leukocytes in human samples also worked well in marmoset blood but failed to provide relevant information in the tissue samples. Confirmation of neutrophil identification was done by nuclear morphology and macrophages were identified by their adherent nature in initial experiments (data not shown). Neutrophils were stained as CD11c dim CD14− and macrophages as CD11c + CD14+ regardless of tissue origin (Figure 1(b) ).", "Neutrophils were stained as CD11c dim CD14− and macrophages as CD11c + CD14+ regardless of tissue origin (Figure 1(b) ). Figure 1 shows the basic division of lymphocytes between T, B, and NK cells from a healthy blood sample. Using this approach, the percentage of NK cells, B-cells, total T-cells, CD8+ T-cells, neutrophils, and monocytes was determined in the blood of naive marmosets (Figure 2 (a), Table 1 ); approximately 63% of all lymphocytes were T cells, 25% B cells, and 12% NK cells.", "Using this approach, the percentage of NK cells, B-cells, total T-cells, CD8+ T-cells, neutrophils, and monocytes was determined in the blood of naive marmosets (Figure 2 (a), Table 1 ); approximately 63% of all lymphocytes were T cells, 25% B cells, and 12% NK cells. The variability of the data is depicted in Figure 2 (a) with the greatest variability observed in the proportion of neutrophils. There were no obvious differences attributable to age or sex of the animals.", "There were no obvious differences attributable to age or sex of the animals. This analysis was also applied to lung and spleen homogenates from naive marmosets (Figures 2(b) and 2(c) ). Greater variability was observed in the data relating to the identification of cell types in tissue samples, attributed to the inherent difficulties in identifying cell types in tissue homogenates by size and granularity and also the smaller cohort of animals.", "Greater variability was observed in the data relating to the identification of cell types in tissue samples, attributed to the inherent difficulties in identifying cell types in tissue homogenates by size and granularity and also the smaller cohort of animals. As expected, low numbers of neutrophils are found in naive spleen or lung tissue (8% both). Healthy mouse spleens typically have approximately 1-2% granulocytes [30] .", "Healthy mouse spleens typically have approximately 1-2% granulocytes [30] . Understandably, there are few reports on the typical cell percentages expected in healthy human individuals for these tissues. However, it is reported that B cells are more prevalent in the spleens of humans at a ratio of 5 to 4 B to T cells than in the lungs which have a ratio of 1 to 8 B to T cells [34] .", "However, it is reported that B cells are more prevalent in the spleens of humans at a ratio of 5 to 4 B to T cells than in the lungs which have a ratio of 1 to 8 B to T cells [34] . In marmoset data reported here, a ratio of 2 to 3 B to T cells in the spleen and 1 to 6 B to T-cells in the lungs was observed compared to a ratio of 3 to 2 B to T cells in mouse spleens [30] . Upon comparison, the marmoset data is generally consistent with previously reported data which is only available for marmoset blood samples [27] and information available for human blood [32, 33] (Table 1 ).", "Upon comparison, the marmoset data is generally consistent with previously reported data which is only available for marmoset blood samples [27] and information available for human blood [32, 33] (Table 1 ). However, one report found the proportion of CD8+ T-cells was almost three times greater in marmosets than humans, 61% to 21% respectively [35] compared to the 30% observed in this study and the work previously reported by Brok et al. [27] .", "[27] . Brok's study involved a small number of animals (eight) and also used a different CD8+ clone to identify cells. Contrastingly, in mice, differences are observed in the proportion of both B cells and neutrophils [31] , although these differences are highly strain specific.", "Contrastingly, in mice, differences are observed in the proportion of both B cells and neutrophils [31] , although these differences are highly strain specific. C57BL/6J mice are reported to have 67% B cells and BALB/C mice 46%; both of which are consistently higher than the percentage found in marmosets and humans of approximately 25% (Table 1 ) [27, 31] . The proportion of neutrophils found in the blood of C57BL/6J mice at 13% is lower than the 35% found in marmosets and the 40-75% expected for healthy human blood.", "The proportion of neutrophils found in the blood of C57BL/6J mice at 13% is lower than the 35% found in marmosets and the 40-75% expected for healthy human blood. This is encouraging as neutrophils play a pivotal role in the innate response to infection [36] . A cross-species comparison suggests that monocytes comprise 3% of leukocytes ( Table 1) .", "A cross-species comparison suggests that monocytes comprise 3% of leukocytes ( Table 1) . Levels of circulating cytokines and chemokines (IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN ) were also quantified in the blood, lung, and spleen of naïve marmosets from the Dstl colony. None of these cytokines were detected in blood samples from uninfected animals; however low levels of MIP-1 , MCP-1, and Rantes were found in spleen and lung tissue.", "None of these cytokines were detected in blood samples from uninfected animals; however low levels of MIP-1 , MCP-1, and Rantes were found in spleen and lung tissue. Preliminary investigation of the immune response has supported the development of marmoset model of infection at Dstl. The levels of different cell types were measured at specific times after challenge with inhalational F. tularensis, B. pseudomallei, and Marburg virus [13] [14] [15] .", "The levels of different cell types were measured at specific times after challenge with inhalational F. tularensis, B. pseudomallei, and Marburg virus [13] [14] [15] . Following challenge with F. tularensis, increasing levels of NK cells, neutrophils, T cells, and macrophages were observed, peaking at 48 hours after challenge before rapidly declining. This study also demonstrated the importance of investigating the immunological response in key target organs, as an increase in CD8+ T cells and T cells was observed in the spleen and lungs but not in the blood.", "This study also demonstrated the importance of investigating the immunological response in key target organs, as an increase in CD8+ T cells and T cells was observed in the spleen and lungs but not in the blood. Increasing levels of various cytokines, MCP-1, MIP-1 , MIP-1 , IL-6, and IL-1 , were observed in Table 1 : Comparison of the percentages of different cell types observed in the blood from healthy marmosets, mice, and humans. Identification markers Marmoset (present data)\n\nMarmoset [27] Mouse 4 [30, 31] Human Asian [32] Human Caucasian [33] Number the lungs, spleen, and blood as the disease progressed (TNF and IFN were not measured in this study).", "Identification markers Marmoset (present data)\n\nMarmoset [27] Mouse 4 [30, 31] Human Asian [32] Human Caucasian [33] Number the lungs, spleen, and blood as the disease progressed (TNF and IFN were not measured in this study). Following inhalational challenge of marmosets with B. pseudomallei, an increase in the number of neutrophils was observed in the blood at 36 hours after challenge, followed by a rapid decline that was associated with an influx of neutrophils into the lung at 46 hours after challenge. A subsequent decline in the number of neutrophils in the lung was associated with the increased number in the spleen of animals that exhibited severe disease and were humanely killed.", "A subsequent decline in the number of neutrophils in the lung was associated with the increased number in the spleen of animals that exhibited severe disease and were humanely killed. There was a gradual increase in the number of macrophages in the spleen as the disease progressed with numbers of macrophages peaking in the blood and lungs at 36 hours after challenge. A rapid decline in the number of macrophages in the lungs and blood was observed by 46 hours after challenge.", "A rapid decline in the number of macrophages in the lungs and blood was observed by 46 hours after challenge. The levels of various cell types and cytokines were also measured in the blood of animals following inhalational challenge with Marburg virus [15] . In these animals a general increase in the numbers of T cells, NK cells, macrophages IFN-, IL-1 , and MCP-1 was observed with time (TNF was not measured).", "In these animals a general increase in the numbers of T cells, NK cells, macrophages IFN-, IL-1 , and MCP-1 was observed with time (TNF was not measured). In order to gain more information from these acute bacterial infection models, we have sought out other markers from the literature. Primarily this was from marmoset models of autoimmune disorders such as rheumatoid arthritis and multiple sclerosis where the cross-reactivity of human antibodies was investigated, as well as the functionality of cells [37] [38] [39] [40] .", "Primarily this was from marmoset models of autoimmune disorders such as rheumatoid arthritis and multiple sclerosis where the cross-reactivity of human antibodies was investigated, as well as the functionality of cells [37] [38] [39] [40] . More recent work at Dstl has reported further cross-reactivity between marmoset cells and human cytokines to induce activity in marmoset T cells [36, 41] . These studies, combined with increasing information available on the cross-reactivity of human antibodies to various NHPs (e.g., NIH NHP reagent resource, has expanded the ability to assess activation markers for disease.", "These studies, combined with increasing information available on the cross-reactivity of human antibodies to various NHPs (e.g., NIH NHP reagent resource, has expanded the ability to assess activation markers for disease. Detection of the following cell surface markers with human antibodies was trialed: CD54 (ICAM-1) associated with cellular adhesion, inflammation, and leukocyte extravasation; CD69 the early activation marker; CD16 as a macrophage activation marker; CD163 the alternative macrophage activation marker; and MHC class II (HLA-DR). CD56 was originally included to identify NK cells; however, it was noted that its expression on T cells was upregulated during disease and that cells defined as CD3+ CD16− CD56+ have been shown to be functionally cytotoxic in marmosets [37, 42] .", "CD56 was originally included to identify NK cells; however, it was noted that its expression on T cells was upregulated during disease and that cells defined as CD3+ CD16− CD56+ have been shown to be functionally cytotoxic in marmosets [37, 42] . These markers have been used to expand on our previously published work to determine changes in the activation status of basic cell types in response to an acute bacterial infection. Animals were challenged with bacteria at a comparable dose either by inhalation ( = 22) or by a systemic route ( = 12) and humanely killed once they had reached a humane endpoint (between day 4 and day 5 after challenge).", "Animals were challenged with bacteria at a comparable dose either by inhalation ( = 22) or by a systemic route ( = 12) and humanely killed once they had reached a humane endpoint (between day 4 and day 5 after challenge). Figure 3 illustrates the cellular activity in representative tissues following inhalational (Figures 3(b) and 3(e)) or systemic challenge (Figures 3(c) and 3(f)) and in naïve samples (Figures 3(a) and 3(d) ). Naïve T and NK cells appear to have similar resting activation states regardless of origin, whereas neutrophils and macrophages have differential expression of activation, for example, CD16.", "Naïve T and NK cells appear to have similar resting activation states regardless of origin, whereas neutrophils and macrophages have differential expression of activation, for example, CD16. In response to disease, the proportions of the cell types appear to remain relativity constant; however, the activation markers provide more detailed information and show involvement of all the cell types explored. Extensive activation was to be expected considering that the samples were taken at the humane endpoint.", "Extensive activation was to be expected considering that the samples were taken at the humane endpoint. There is also extensive variation between The response to infection within the lungs has similarities across disease routes in terms of neutrophil reduced expression of CD16 and CD54 and macrophage increased expression of CD16 and reduction in MHCII. Unexpectedly, the T and NK cells appear to be more actively involved in systemic disease, indicating that the disease develops a pneumonic element regardless of initial route of infection.", "Unexpectedly, the T and NK cells appear to be more actively involved in systemic disease, indicating that the disease develops a pneumonic element regardless of initial route of infection. Levels of circulating cytokines and chemokines (IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN ) were also quantified in the lung and spleen samples. All of the cytokines (with the exception of Rantes) were expressed at high levels (ng/mg) in all samples, which was expected as the animals had succumbed to terminal disease.", "All of the cytokines (with the exception of Rantes) were expressed at high levels (ng/mg) in all samples, which was expected as the animals had succumbed to terminal disease. The work presented here adds significant relevant information to the marmoset models of infection and to the understanding of the immune response in these animals. This work extends marmoset immunology from autoimmune disorders into the field of infectious diseases; this coupled with an increase in the information available on crossreactivity of human reagents to a variety of NHPs increases the utility/application of marmosets as models of human disease.", "This work extends marmoset immunology from autoimmune disorders into the field of infectious diseases; this coupled with an increase in the information available on crossreactivity of human reagents to a variety of NHPs increases the utility/application of marmosets as models of human disease. In conclusion, the immune response in marmosets to infectious disease can be characterised in terms of the phenotype and activation status of all the major immune cells and key cytokine and chemokine expression. This can aid in the identification of correlates of infection or protection in medical countermeasures assessment studies.", "This can aid in the identification of correlates of infection or protection in medical countermeasures assessment studies. This information can also potentially be used for pivotal studies to support licensure of products under the FDA Animal Rule. This, in conjunction with the small size of marmosets, their immune response to infection that is comparable to humans, and the ability to house more statistically relevant numbers within high containment, makes the marmoset an appropriate animal model for biodefense-related pathogens." ]

[ "The common marmoset (Callithrix jacchus) is increasingly being utilised as a nonhuman primate model for human disease, ranging from autoimmune to infectious disease. In order to fully exploit these models, meaningful comparison to the human host response is necessary. Commercially available reagents, primarily targeted to human cells, were utilised to assess the phenotype and activation status of key immune cell types and cytokines in naive and infected animals.", "Commercially available reagents, primarily targeted to human cells, were utilised to assess the phenotype and activation status of key immune cell types and cytokines in naive and infected animals. Single cell suspensions of blood, spleen, and lung were examined. Generally, the phenotype of cells was comparable between humans and marmosets, with approximately 63% of all lymphocytes in the blood of marmosets being T cells, 25% B-cells, and 12% NK cells.", "Generally, the phenotype of cells was comparable between humans and marmosets, with approximately 63% of all lymphocytes in the blood of marmosets being T cells, 25% B-cells, and 12% NK cells. The percentage of neutrophils in marmoset blood were more similar to human values than mouse values. Comparison of the activation status of cells following experimental systemic or inhalational infection exhibited different trends in different tissues, most obvious in cell types active in the innate immune response.", "Comparison of the activation status of cells following experimental systemic or inhalational infection exhibited different trends in different tissues, most obvious in cell types active in the innate immune response. This work significantly enhances the ability to understand the immune response in these animals and fortifies their use as models of infectious disease. Text: The common marmoset (Callithrix jacchus), a New World monkey (NWM) species is a small, arboreal nonhuman primate (NHP), native to the Atlantic Coastal Forest in Northeast Brazil and parts of South East Brazil.", "Text: The common marmoset (Callithrix jacchus), a New World monkey (NWM) species is a small, arboreal nonhuman primate (NHP), native to the Atlantic Coastal Forest in Northeast Brazil and parts of South East Brazil. In recent years the common marmoset has become more widely used in applied biomedical research, and an increasing body of evidence suggests the physiological and immunological responses to biological insults are similar between marmosets and humans [1] . In the field of infectious disease, the marmoset is primarily being investigated as an alternative NHP model to complement the more traditionally used Old World monkeys (OWM) (e.g., rhesus and cynomolgus macaques).", "In the field of infectious disease, the marmoset is primarily being investigated as an alternative NHP model to complement the more traditionally used Old World monkeys (OWM) (e.g., rhesus and cynomolgus macaques). Evolutionarily, both NWM and OWM sit within the simiiformes infraorder of the suborder Haplorhini of primates [2] . Marmosets sit within the family Callitrichidae of the Platyrrhini parvorder, while OWM sit within the Cercopithecidae family of the Catarrhini Parvorder.", "Marmosets sit within the family Callitrichidae of the Platyrrhini parvorder, while OWM sit within the Cercopithecidae family of the Catarrhini Parvorder. Marmosets therefore are separated from Old World monkeys by one ancestral step and are a lower order primate. Marmosets have been used to model the infection syndrome caused by a number of public health pathogens including Lassa virus [3] , Hepatitis C virus [4] , Dengue virus [5] , Herpesvirus [6] , Junin virus [7] Rift Valley Fever [8] , and SARS [9] .", "Marmosets have been used to model the infection syndrome caused by a number of public health pathogens including Lassa virus [3] , Hepatitis C virus [4] , Dengue virus [5] , Herpesvirus [6] , Junin virus [7] Rift Valley Fever [8] , and SARS [9] . Marmosets have also been used to model a number of biodefense pathogens including Eastern Equine Encephalitis virus [10] , Bacillus anthracis [11] , Francisella tularensis [12, 13] , Burkholderia pseudomallei [14] , Marburg haemorrhagic fever virus [15, 16] , Ebola haemorrhagic fever virus [16] , and Variola virus [17] . The utility of marmosets to assess medical countermeasures has also been demonstrated; a vaccine has been tested for Lassa fever [18] and the efficacy of ciprofloxacin and levofloxacin has been tested as postexposure therapies for anthrax and tularemia, respectively [19, 20] .", "The utility of marmosets to assess medical countermeasures has also been demonstrated; a vaccine has been tested for Lassa fever [18] and the efficacy of ciprofloxacin and levofloxacin has been tested as postexposure therapies for anthrax and tularemia, respectively [19, 20] . In order to exploit these models fully and to allow meaningful comparison with the human condition, the response of the immune system to infection/therapy needs to be 2 Journal of Immunology Research characterised and understood. Generally, NHPs have a close molecular, immunological, reproductive, and neurological similarity with humans making them ideal surrogates for humans and the study of infectious diseases.", "Generally, NHPs have a close molecular, immunological, reproductive, and neurological similarity with humans making them ideal surrogates for humans and the study of infectious diseases. There is a high level of gene homology between humans and NHPs which underlies physiological and biochemical similarities. Similarities at the genetic level extend to the phenotypical level making NHPs well suited to modelling pathophysiological responses in man [21] .", "Similarities at the genetic level extend to the phenotypical level making NHPs well suited to modelling pathophysiological responses in man [21] . Immunologically, there is a high degree of homology between humans and marmosets [22] . The similarity of various immunological factors produced by humans and marmosets has been investigated at both the genetic and protein levels.", "The similarity of various immunological factors produced by humans and marmosets has been investigated at both the genetic and protein levels. There is at least 95% homology between human costimulatory molecules (e.g., CD80, CD86 etc.) and those of marmosets [23] .", "and those of marmosets [23] . Also the immunoglobulin and T-cell receptor repertoire of humans and marmosets show at least 80% homology [24, 25] . Currently, the availability of commercial reagents specifically designed for the marmoset is limited although a number of antibodies designed for use with human samples have been shown to cross-react with leucocytes from marmoset blood [26] [27] [28] .", "Currently, the availability of commercial reagents specifically designed for the marmoset is limited although a number of antibodies designed for use with human samples have been shown to cross-react with leucocytes from marmoset blood [26] [27] [28] . However, these reagents have not been exploited to investigate the immune response to infectious disease. To date, investigation of the immune response in marmosets has primarily been achieved using pathogen-specific antibodies to determine the serological response using ELISA such as in the smallpox, Dengue, Rift Valley Fever, and Herpes models [5, 6, 8, 17] or by immunohistochemistry to identify, for example, CD8+, CD3+, CD20+ cells, and IL-6 in the smallpox model [17] ; neutrophils and macrophages in the Herpes model [6] ; or CD3+ and CD20+ cells in the Lassa model [3] .", "To date, investigation of the immune response in marmosets has primarily been achieved using pathogen-specific antibodies to determine the serological response using ELISA such as in the smallpox, Dengue, Rift Valley Fever, and Herpes models [5, 6, 8, 17] or by immunohistochemistry to identify, for example, CD8+, CD3+, CD20+ cells, and IL-6 in the smallpox model [17] ; neutrophils and macrophages in the Herpes model [6] ; or CD3+ and CD20+ cells in the Lassa model [3] . The work presented here focuses on understanding the immune profile of the naive marmoset as well as identifying and quantifying the immune response to infectious disease. The aim of this work is to determine key changes and identify correlates of infection or protection.", "The aim of this work is to determine key changes and identify correlates of infection or protection. Healthy sexually mature common marmosets (C. jacchus) were obtained from the Dstl Porton Down breeding colony and housed in vasectomized male and female pairs. The Dstl colony was established during the 1970s and is a closed colony with a stable genotype.", "The Dstl colony was established during the 1970s and is a closed colony with a stable genotype. Animals included in these studies were mixed sex pairs, between 18 months and 5 years old and weighing between 320 g to 500 g. All animals were allowed free access to food and water as well as environmental enrichment. All animal studies were carried out in accordance with the UK Animals (Scientific Procedures) Act of 1986 and the Codes of Practice for the Housing and Care of Animals used in Scientific Procedures 1989.", "All animal studies were carried out in accordance with the UK Animals (Scientific Procedures) Act of 1986 and the Codes of Practice for the Housing and Care of Animals used in Scientific Procedures 1989. Animals were challenged with an intracellular pathogen by either the subcutaneous or inhalational route and were humanely killed at various time points after challenge. Prior to the infection study, animals were bled to determine baseline immunological parameters.", "Prior to the infection study, animals were bled to determine baseline immunological parameters. Studies were performed to establish infection models in order to evaluate the efficacy of suitable therapies for transition ultimately to the clinic. Populations. Blood and tissue samples were homogenised to provide single cell suspensions [12] .", "Blood and tissue samples were homogenised to provide single cell suspensions [12] . Red blood cells were lysed, and the mixed leucocyte population was washed and stained with various combinations of the following fluorescent antibody stains: CD3 (SP34-2), CD8 (LT8), CD11c (SHCL3), CD14 (M5E2), CD16 (3G8), CD20 (Bly1), CD45RA (5H9), CD54 (HCD54), CD56 (B159), CD69 (FN50), CD163 (GHI/61), and MCHII (L243) (BD Bioscience, Insight Bioscience, AbD serotec). Samples were fixed in 4% paraformaldehyde for 48 hrs at 4 ∘ C and analysed by flow cytometry (FACScanto II BD) within 72 hours of staining.", "Samples were fixed in 4% paraformaldehyde for 48 hrs at 4 ∘ C and analysed by flow cytometry (FACScanto II BD) within 72 hours of staining. Levels of circulating cytokines and chemokines were also quantified in the blood of marmosets from the Dstl colony using human multiplex kits available commercially (BD cytokine flex beads and the Luminex system). These systems show significant cross-reactivity with the marmoset suggesting a high degree of conservation between the two species for IL-6, MIP-1 , MIP-1 , and MCP-1 [29] .", "These systems show significant cross-reactivity with the marmoset suggesting a high degree of conservation between the two species for IL-6, MIP-1 , MIP-1 , and MCP-1 [29] . However, for other cytokines that are pivotal in the innate response, TNF and IFN reagents were obtained from U-CyTech Biosciences and Mabtech AB, respectively, due to a lack of cross-reactivity observed within the kit obtained from BD [13] . In order to fully characterise the immune response to infectious agent in the marmoset, single cell suspensions of lung and spleen tissue were also examined in conjunction with the traditionally used blood cells.", "In order to fully characterise the immune response to infectious agent in the marmoset, single cell suspensions of lung and spleen tissue were also examined in conjunction with the traditionally used blood cells. These tissue homogenates are of particular interest in relation to target sites of infection: the lung as the site of initial infection following an inhalational challenge and the spleen as a representative organ following a parental challenge. Cell types targeted during this analysis include cells important in the innate response (e.g., neutrophils, macrophages, and NK cells) and the adaptive response (T and B cells) with a view to determine the response to infection and vaccination and to derive immune correlates of infection/protection.", "Cell types targeted during this analysis include cells important in the innate response (e.g., neutrophils, macrophages, and NK cells) and the adaptive response (T and B cells) with a view to determine the response to infection and vaccination and to derive immune correlates of infection/protection. Dapi was included as a nuclear marker to ensure that the initial gating included only intact cells. Basic cell types in blood were easily identified by measuring size (forward) and granularity (side) scatter (Figure 1(a) ).", "Basic cell types in blood were easily identified by measuring size (forward) and granularity (side) scatter (Figure 1(a) ). Identification of cell types in tissue samples was more difficult as the scatter profiles are less clearly compartmentalized. The common leukocyte antigen (CD45) normally used to locate all leukocytes in human samples also worked well in marmoset blood but failed to provide relevant information in the tissue samples.", "The common leukocyte antigen (CD45) normally used to locate all leukocytes in human samples also worked well in marmoset blood but failed to provide relevant information in the tissue samples. Confirmation of neutrophil identification was done by nuclear morphology and macrophages were identified by their adherent nature in initial experiments (data not shown). Neutrophils were stained as CD11c dim CD14− and macrophages as CD11c + CD14+ regardless of tissue origin (Figure 1(b) ).", "Neutrophils were stained as CD11c dim CD14− and macrophages as CD11c + CD14+ regardless of tissue origin (Figure 1(b) ). Figure 1 shows the basic division of lymphocytes between T, B, and NK cells from a healthy blood sample. Using this approach, the percentage of NK cells, B-cells, total T-cells, CD8+ T-cells, neutrophils, and monocytes was determined in the blood of naive marmosets (Figure 2 (a), Table 1 ); approximately 63% of all lymphocytes were T cells, 25% B cells, and 12% NK cells.", "Using this approach, the percentage of NK cells, B-cells, total T-cells, CD8+ T-cells, neutrophils, and monocytes was determined in the blood of naive marmosets (Figure 2 (a), Table 1 ); approximately 63% of all lymphocytes were T cells, 25% B cells, and 12% NK cells. The variability of the data is depicted in Figure 2 (a) with the greatest variability observed in the proportion of neutrophils. There were no obvious differences attributable to age or sex of the animals.", "There were no obvious differences attributable to age or sex of the animals. This analysis was also applied to lung and spleen homogenates from naive marmosets (Figures 2(b) and 2(c) ). Greater variability was observed in the data relating to the identification of cell types in tissue samples, attributed to the inherent difficulties in identifying cell types in tissue homogenates by size and granularity and also the smaller cohort of animals.", "Greater variability was observed in the data relating to the identification of cell types in tissue samples, attributed to the inherent difficulties in identifying cell types in tissue homogenates by size and granularity and also the smaller cohort of animals. As expected, low numbers of neutrophils are found in naive spleen or lung tissue (8% both). Healthy mouse spleens typically have approximately 1-2% granulocytes [30] .", "Healthy mouse spleens typically have approximately 1-2% granulocytes [30] . Understandably, there are few reports on the typical cell percentages expected in healthy human individuals for these tissues. However, it is reported that B cells are more prevalent in the spleens of humans at a ratio of 5 to 4 B to T cells than in the lungs which have a ratio of 1 to 8 B to T cells [34] .", "However, it is reported that B cells are more prevalent in the spleens of humans at a ratio of 5 to 4 B to T cells than in the lungs which have a ratio of 1 to 8 B to T cells [34] . In marmoset data reported here, a ratio of 2 to 3 B to T cells in the spleen and 1 to 6 B to T-cells in the lungs was observed compared to a ratio of 3 to 2 B to T cells in mouse spleens [30] . Upon comparison, the marmoset data is generally consistent with previously reported data which is only available for marmoset blood samples [27] and information available for human blood [32, 33] (Table 1 ).", "Upon comparison, the marmoset data is generally consistent with previously reported data which is only available for marmoset blood samples [27] and information available for human blood [32, 33] (Table 1 ). However, one report found the proportion of CD8+ T-cells was almost three times greater in marmosets than humans, 61% to 21% respectively [35] compared to the 30% observed in this study and the work previously reported by Brok et al. [27] .", "[27] . Brok's study involved a small number of animals (eight) and also used a different CD8+ clone to identify cells. Contrastingly, in mice, differences are observed in the proportion of both B cells and neutrophils [31] , although these differences are highly strain specific.", "Contrastingly, in mice, differences are observed in the proportion of both B cells and neutrophils [31] , although these differences are highly strain specific. C57BL/6J mice are reported to have 67% B cells and BALB/C mice 46%; both of which are consistently higher than the percentage found in marmosets and humans of approximately 25% (Table 1 ) [27, 31] . The proportion of neutrophils found in the blood of C57BL/6J mice at 13% is lower than the 35% found in marmosets and the 40-75% expected for healthy human blood.", "The proportion of neutrophils found in the blood of C57BL/6J mice at 13% is lower than the 35% found in marmosets and the 40-75% expected for healthy human blood. This is encouraging as neutrophils play a pivotal role in the innate response to infection [36] . A cross-species comparison suggests that monocytes comprise 3% of leukocytes ( Table 1) .", "A cross-species comparison suggests that monocytes comprise 3% of leukocytes ( Table 1) . Levels of circulating cytokines and chemokines (IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN ) were also quantified in the blood, lung, and spleen of naïve marmosets from the Dstl colony. None of these cytokines were detected in blood samples from uninfected animals; however low levels of MIP-1 , MCP-1, and Rantes were found in spleen and lung tissue.", "None of these cytokines were detected in blood samples from uninfected animals; however low levels of MIP-1 , MCP-1, and Rantes were found in spleen and lung tissue. Preliminary investigation of the immune response has supported the development of marmoset model of infection at Dstl. The levels of different cell types were measured at specific times after challenge with inhalational F. tularensis, B. pseudomallei, and Marburg virus [13] [14] [15] .", "The levels of different cell types were measured at specific times after challenge with inhalational F. tularensis, B. pseudomallei, and Marburg virus [13] [14] [15] . Following challenge with F. tularensis, increasing levels of NK cells, neutrophils, T cells, and macrophages were observed, peaking at 48 hours after challenge before rapidly declining. This study also demonstrated the importance of investigating the immunological response in key target organs, as an increase in CD8+ T cells and T cells was observed in the spleen and lungs but not in the blood.", "This study also demonstrated the importance of investigating the immunological response in key target organs, as an increase in CD8+ T cells and T cells was observed in the spleen and lungs but not in the blood. Increasing levels of various cytokines, MCP-1, MIP-1 , MIP-1 , IL-6, and IL-1 , were observed in Table 1 : Comparison of the percentages of different cell types observed in the blood from healthy marmosets, mice, and humans. Identification markers Marmoset (present data)\n\nMarmoset [27] Mouse 4 [30, 31] Human Asian [32] Human Caucasian [33] Number the lungs, spleen, and blood as the disease progressed (TNF and IFN were not measured in this study).", "Identification markers Marmoset (present data)\n\nMarmoset [27] Mouse 4 [30, 31] Human Asian [32] Human Caucasian [33] Number the lungs, spleen, and blood as the disease progressed (TNF and IFN were not measured in this study). Following inhalational challenge of marmosets with B. pseudomallei, an increase in the number of neutrophils was observed in the blood at 36 hours after challenge, followed by a rapid decline that was associated with an influx of neutrophils into the lung at 46 hours after challenge. A subsequent decline in the number of neutrophils in the lung was associated with the increased number in the spleen of animals that exhibited severe disease and were humanely killed.", "A subsequent decline in the number of neutrophils in the lung was associated with the increased number in the spleen of animals that exhibited severe disease and were humanely killed. There was a gradual increase in the number of macrophages in the spleen as the disease progressed with numbers of macrophages peaking in the blood and lungs at 36 hours after challenge. A rapid decline in the number of macrophages in the lungs and blood was observed by 46 hours after challenge.", "A rapid decline in the number of macrophages in the lungs and blood was observed by 46 hours after challenge. The levels of various cell types and cytokines were also measured in the blood of animals following inhalational challenge with Marburg virus [15] . In these animals a general increase in the numbers of T cells, NK cells, macrophages IFN-, IL-1 , and MCP-1 was observed with time (TNF was not measured).", "In these animals a general increase in the numbers of T cells, NK cells, macrophages IFN-, IL-1 , and MCP-1 was observed with time (TNF was not measured). In order to gain more information from these acute bacterial infection models, we have sought out other markers from the literature. Primarily this was from marmoset models of autoimmune disorders such as rheumatoid arthritis and multiple sclerosis where the cross-reactivity of human antibodies was investigated, as well as the functionality of cells [37] [38] [39] [40] .", "Primarily this was from marmoset models of autoimmune disorders such as rheumatoid arthritis and multiple sclerosis where the cross-reactivity of human antibodies was investigated, as well as the functionality of cells [37] [38] [39] [40] . More recent work at Dstl has reported further cross-reactivity between marmoset cells and human cytokines to induce activity in marmoset T cells [36, 41] . These studies, combined with increasing information available on the cross-reactivity of human antibodies to various NHPs (e.g., NIH NHP reagent resource, has expanded the ability to assess activation markers for disease.", "These studies, combined with increasing information available on the cross-reactivity of human antibodies to various NHPs (e.g., NIH NHP reagent resource, has expanded the ability to assess activation markers for disease. Detection of the following cell surface markers with human antibodies was trialed: CD54 (ICAM-1) associated with cellular adhesion, inflammation, and leukocyte extravasation; CD69 the early activation marker; CD16 as a macrophage activation marker; CD163 the alternative macrophage activation marker; and MHC class II (HLA-DR). CD56 was originally included to identify NK cells; however, it was noted that its expression on T cells was upregulated during disease and that cells defined as CD3+ CD16− CD56+ have been shown to be functionally cytotoxic in marmosets [37, 42] .", "CD56 was originally included to identify NK cells; however, it was noted that its expression on T cells was upregulated during disease and that cells defined as CD3+ CD16− CD56+ have been shown to be functionally cytotoxic in marmosets [37, 42] . These markers have been used to expand on our previously published work to determine changes in the activation status of basic cell types in response to an acute bacterial infection. Animals were challenged with bacteria at a comparable dose either by inhalation ( = 22) or by a systemic route ( = 12) and humanely killed once they had reached a humane endpoint (between day 4 and day 5 after challenge).", "Animals were challenged with bacteria at a comparable dose either by inhalation ( = 22) or by a systemic route ( = 12) and humanely killed once they had reached a humane endpoint (between day 4 and day 5 after challenge). Figure 3 illustrates the cellular activity in representative tissues following inhalational (Figures 3(b) and 3(e)) or systemic challenge (Figures 3(c) and 3(f)) and in naïve samples (Figures 3(a) and 3(d) ). Naïve T and NK cells appear to have similar resting activation states regardless of origin, whereas neutrophils and macrophages have differential expression of activation, for example, CD16.", "Naïve T and NK cells appear to have similar resting activation states regardless of origin, whereas neutrophils and macrophages have differential expression of activation, for example, CD16. In response to disease, the proportions of the cell types appear to remain relativity constant; however, the activation markers provide more detailed information and show involvement of all the cell types explored. Extensive activation was to be expected considering that the samples were taken at the humane endpoint.", "Extensive activation was to be expected considering that the samples were taken at the humane endpoint. There is also extensive variation between The response to infection within the lungs has similarities across disease routes in terms of neutrophil reduced expression of CD16 and CD54 and macrophage increased expression of CD16 and reduction in MHCII. Unexpectedly, the T and NK cells appear to be more actively involved in systemic disease, indicating that the disease develops a pneumonic element regardless of initial route of infection.", "Unexpectedly, the T and NK cells appear to be more actively involved in systemic disease, indicating that the disease develops a pneumonic element regardless of initial route of infection. Levels of circulating cytokines and chemokines (IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN ) were also quantified in the lung and spleen samples. All of the cytokines (with the exception of Rantes) were expressed at high levels (ng/mg) in all samples, which was expected as the animals had succumbed to terminal disease.", "All of the cytokines (with the exception of Rantes) were expressed at high levels (ng/mg) in all samples, which was expected as the animals had succumbed to terminal disease. The work presented here adds significant relevant information to the marmoset models of infection and to the understanding of the immune response in these animals. This work extends marmoset immunology from autoimmune disorders into the field of infectious diseases; this coupled with an increase in the information available on crossreactivity of human reagents to a variety of NHPs increases the utility/application of marmosets as models of human disease.", "This work extends marmoset immunology from autoimmune disorders into the field of infectious diseases; this coupled with an increase in the information available on crossreactivity of human reagents to a variety of NHPs increases the utility/application of marmosets as models of human disease. In conclusion, the immune response in marmosets to infectious disease can be characterised in terms of the phenotype and activation status of all the major immune cells and key cytokine and chemokine expression. This can aid in the identification of correlates of infection or protection in medical countermeasures assessment studies.", "This can aid in the identification of correlates of infection or protection in medical countermeasures assessment studies. This information can also potentially be used for pivotal studies to support licensure of products under the FDA Animal Rule. This, in conjunction with the small size of marmosets, their immune response to infection that is comparable to humans, and the ability to house more statistically relevant numbers within high containment, makes the marmoset an appropriate animal model for biodefense-related pathogens." ]

[ "Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction.", "Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries.", "The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil.", "Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity. Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs).", "Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs). These antivirals have significantly increased response rates (up to 98%) and greatly reduced treatment duration [1] . Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] .", "Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] . However, the extreme mutation and high replication rates of HCV, together with the immune system pressure, lead to a remarkable genetic variability that can compromise the high response rates to DAAs due to the preexistence of resistanceassociated substitutions (RASs) [5, 6] . Each drug or class of DAA is characterized by specific resistance profiles.", "Each drug or class of DAA is characterized by specific resistance profiles. The likelihood that a DAA will select for and allow outgrowth of viral populations carrying RASs depends on the DAA's genetic barrier to resistance (the number and type of mutations needed to generate an amino acid substitution that confers resistance), the viral fitness (replicative capacity) of the resistant variant, and viral genotypes and subtypes [7, 8] . The prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] .", "The prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] . However, apart from Brazil and Argentina, this issue has not been fully addressed in other South American countries yet [9, [17] [18] [19] . The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] .", "The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] . In this study, we explored the presence of resistance variants to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients chronically infected with hepatitis C. Here, we aimed to contribute to the knowledge of the circulation of HCV resistant variants in the South American region. Samples.", "Samples. Serum samples were obtained from 31 patients with serological markers for HCV, which were recruited between 2015 and 2017 at the Gastroenterology Clinic from Hospital de Clínicas, Montevideo, Uruguay. HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA).", "HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA). Patients selected for this study were both chronically infected with HCV genotype 1 and DAA treatment-naïve at the time of blood extraction. Written informed consent was obtained from all patients.", "Written informed consent was obtained from all patients. The studies have been performed according to the World Medical Association Declaration of Helsinki and approved by the appropriate institutional board (Hospital de Clínicas ethical committee). 2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification.", "2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification. Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol.", "Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol. The viral RNA was heated at 65°C for 5 min and used as a template for a reverse transcription reaction. The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA).", "The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA). The reverse transcription was performed at 42°C for 50 min, and then the reverse transcriptase enzyme was inactivated at 70°C for 15 min. PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously described [10] .", "PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously described [10] . Amplicons were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Science, Buckinghamshire, UK) according to the manufacturer's protocol. 2.3. NS5A and NS5B Sequencing.", "2.3. NS5A and NS5B Sequencing. The purified product was then sequenced using the same sets of primers used for PCR amplification. Bidirectional Sanger sequencing was performed by Macrogen Korea (\n\n2.4. NS5A and NS5B Genotype Determination.", "NS5A and NS5B Genotype Determination. HCV NS5A and NS5B consensus sequences obtained from Uruguayan patients were aligned with sequences from HCV representing all genotypes and main subtypes isolated in different geographic regions of the world. These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] .", "These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] . For strains included in these studies, see Supplementary Material Table S1 . Sequences were aligned using the CLUSTAL W software [23] .", "Sequences were aligned using the CLUSTAL W software [23] . Once aligned, the best evolutionary model that described our sequence data was assessed using ModelGenerator program [24] . Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] .", "Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] . For NS5A, 953 nucleotides (positions 6367 to 7319, relative to HCV 1a reference strain, H77 NC_004102) were included in the phylogenetic analysis, whereas for NS5B, only 361 nucleotides corresponding to the Okamoto region (positions 8265 to 8625, relative to strain H77 NC_004102) were included. As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates).", "As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates). For NS5A 1a Uruguayan sequences (n = 20), a second alignment and maximum likelihood phylogenetic tree was generated in order to analyze HCV evolutionary relationships between Uruguayan, Brazilian, and worldwide strains. For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2.", "For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2. 2.5. NS5A and NS5B Sequence Analysis.", "2.5. NS5A and NS5B Sequence Analysis. In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide.", "In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide. For this purpose, NS5A gene sequences corresponding to subtypes 1a (n = 160) and 1b (n = 88) were retrieved from Los Alamos HCV sequence database and from the NIAID ViPR [21, 22] . Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively.", "Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively. Using Seqman program, implemented in DNAStar 5.01 package (DNASTAR, Madison, USA), a world consensus nucleotide sequences were generated for each gene and subtype. Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated.", "Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated. The amino acid sequences obtained were compared in order to explore the presence of RASs as well as the presence of polymorphisms at a RAS position (RAPs) in Uruguayan HCV strains. RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] .", "RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] . To study the genetic variability of NS5A and NS5B regions of HCV strains circulating in Uruguayan patients, sequences of these regions (accession numbers MH070029-MH070090) were aligned with corresponding sequences from 59 HCV strains isolated elsewhere, representing all genotypes and main subtypes (for strains included in these analyses, see Supplementary Material Table S1 ). Therefore, maximum likelihood phylogenetic trees were constructed.", "Therefore, maximum likelihood phylogenetic trees were constructed. The results of these studies are shown in Figure 1 All strains in the phylogenies were assigned according to their genotype, and each cluster was supported by very high bootstrap values for both analyzed regions. Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b.", "Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b. The results of NS5A (Figure 1 (a)) and NS5B (Figure 1 \n\nGenotype 1b phylogenetic analyses were concordant for both genomic regions in all 31 sequences, suggesting no recombination events between these regions. To further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ).", "To further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ). As was previously described, two distinct 1a clades (clades 1 and 2) were observed. Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] .", "Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] . Whereas NS5A Uruguayan strains (in red) did not cluster in a particular clade, rather, they grouped dispersedly within all major world clades. With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence).", "With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence). AA substitutions at positions previously found to be potentially associated with resistance to NS5A inhibitors, as well as polymorphisms at a RAS position, were identified. These results are summarized in Table 1 .", "These results are summarized in Table 1 . RASs to NS5A inhibitors (L31M and L31V) were identified in 2 strains out of 25 (8%) fully sequenced samples. RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q.", "RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q. Although these substitutions were not reported as resistant, some changes at these positions were previously described as RASs in subtype 1a, namely H58D and K24R [27, 28] . Finally, substitution E62D was found in one subtype 1a strain.", "Finally, substitution E62D was found in one subtype 1a strain. This change is considered as a secondary substitution because, although it does not confer resistance by itself, when combined with a known RAS it does. In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] .", "In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] . In addition, several polymorphisms that have not been previously reported to be associated with a resistant phenotype were also detected (see Supplementary Material Table S3 ). In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences.", "In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences. Almost full-length AA sequences were obtained in 26 out of 31 analyzed strains. 23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein.", "23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein. This issue limited our studies, since many of the described RASs are observed as of residue 553. Importantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%).", "Importantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%). C451R was found in two isolates while A421V was found in only one. In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed.", "In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed. Finally, we found two RAPs: A421V (in 2 subtype 1b strains) and A553G (in 1 subtype 1a strain). Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] .", "Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] . In position 553, the substitution reported as resistant was A553T [8] . As was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ).", "As was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ). The advent of DAAs therapies constitutes one of the major breakthroughs in HCV infected patients management. However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems.", "However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems. The main limiting factors for worldwide access to DAAs in our region concern the high cost, the inadequate management of public healthcare systems, the limited access of low-income or uninsured populations to healthcare providers, and the lack of accurate epidemiological information [20, [30] [31] [32] . In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases.", "In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases. Despite the high rates of viral response achieved with DAA-based treatments, still 1 to10% of the patients fails to eliminate infection, and in these cases, baseline and emergent resistance variants turn out to be key factors contributing to treatment failure [5, 17, 33] . Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating.", "Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating. These facts could compromise the effectiveness of these new therapies in our country. We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] .", "We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] . The present study describes the prevalence of baseline NS5A and NS5B RASs in HCV genotype 1 infected DAA-naïve patients in a Uruguayan cohort. The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] .", "The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] . However, NS5A sequences from South America are poorly analyzed yet [9, 44] . Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] .", "Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] . Importantly, the prevalence and type of baseline NS5A RASs varies slightly by geographic regions. For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] .", "For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] . For this reason, we believe that there is a need to contribute data from our region, for which we still do not have enough information, apart from Brazil [9, 44] . The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ).", "The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ). L31M substitution confers resistance to daclatasvir (DCV), ledipasvir (LDV), and elbasvir (EBV) in both 1a and 1b subtypes [5, 6, 8, 28, 47, 48] , whereas substitution L31V does it to DCV in subtypes 1a and 1b, to LDV in subtype 1b, and to EBV in subtype 1a [5, 6, 28] . Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] .", "Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] . The substitutions H58P and K24Q found in two patients are considered as resistance-associated polymorphisms (RAPs). The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] .", "The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] . The substitution H58P was found as a baseline RAP in relapsers to LDV (HARVONI prescription, media/files/pdfs/medicines/liver-disease/harvoni/harvoni_pi. pdf?la=en).", "pdf?la=en). However, it is sometimes regarded as a RAS [10, 51] , despite conferring only 1.2 fold change in resistance in in vitro studies using the 1a replicon system [39] . We did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] .", "We did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] . The amino acid substitution E62H was found in one Uruguayan patient. Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] .", "Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] . The presence of baseline NS5A RASs impacts treatment outcome in some patient groups by affecting SVR rates. The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use.", "The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use. Regarding NS5B gene, global analysis (with the exception of South America [17, 19] ) revealed that NS5B DAA resistance substitutions are infrequent [14] . Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%).", "Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%). Substitutions found in this work were A421V and S556G associated in subtype 1a with resistance to BCV and dasabuvir (DSV), respectively [8, 28, 29, 54, 55] , and Q556R associated with resistance to DSV both in genotype 1a and 1b [12, 28] . Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV.", "Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV. In these cases, it appeared in combination with G558R (Trial Coral I-Cohort 2: RAPs in positions 421 and 553 (A421V in two subtype 1b isolates and A553G in one subtype 1b isolate) were also found. Although A421V has been associated with resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] .", "Although A421V has been associated with resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] . In position 553, the substitutions reported as resistant are A553T in subtype 1a [8] and A553V in subtype 1b [54] , conferring resistance to DSV. In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ).", "In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ). Nevertheless, substitution A421V was found in Brazil [17] , Argentina [19] , and Uruguay. The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] .", "The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] . Our findings further confirm and complement previous studies which evidenced a low prevalence of this substitution in vivo, probably due to its low replicative fitness [14, 18, 57] . Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] .", "Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] . These results show both diversity in the baseline polymorphisms found in different Latin American countries and in the evolutionary relationships of Uruguayan isolates ( Figure 2 ). This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host.", "This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host. It is worth mentioning that we live in a vast continent inhabited by populations with different genotypic characteristics that might, depending on the situation, require different approaches to treatment. Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] .", "Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] . Altogether, we believe that it could be important to carry out studies throughout the South American region in order to establish the prevalence of RASs in NS5A and NS5B in different countries. In fact, this will aid in understanding that not every treatment regimen might be adequate for every patient and country.", "In fact, this will aid in understanding that not every treatment regimen might be adequate for every patient and country. The data we presented here might guide not only physicians in making therapeutic decisions but also public health authorities in approving more diverse treatment combinations. These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population.", "These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population. To our knowledge, the present study revealed for the first time the presence of RASs in the NS5A and NS5B regions of HCV genotype 1 Uruguayan strains from patients who have not been previously treated with DAAs and is one of the few South American countries to report on this matter. It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure.", "It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure. However, individualized DAA therapy based on baseline resistance analysis may be beneficial for optimizing treatment efficacy in patients with HCV genotype 1 infection and risk factors for treatment failure. Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions.", "Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions. The data used to support the findings of this study are included within the article. The authors declare that they have no conflicts of interest. Fabián Aldunate and Natalia Echeverría contributed equally to this work.", "Fabián Aldunate and Natalia Echeverría contributed equally to this work. Supplementary Material Table S1 : hepatitis C Virus NS5A and NS5B sequences used as representatives of each genotype to perform the phylogenetic analysis. Their corresponding genotype, country of isolation, and GenBank accession number are indicated.", "Their corresponding genotype, country of isolation, and GenBank accession number are indicated. Supplementary Material Table S2 : hepatitis C Virus NS5A subtype 1a sequences used to reveal evolutionary relationships between Uruguayan strains and others isolated elsewhere. Their corresponding country of isolation and GenBank accession number are indicated.", "Their corresponding country of isolation and GenBank accession number are indicated. Supplementary Material Table S3 : amino acid substitutions in NS5A protein not previously associated with resistance to NS5A inhibitors. Supplementary Material Table S4 : amino acid substitutions in NS5B protein not previously associated with resistance to polymerase inhibitors. (Supplementary Materials)" ]

[ "Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction.", "Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries.", "The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil.", "Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity. Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs).", "Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs). These antivirals have significantly increased response rates (up to 98%) and greatly reduced treatment duration [1] . Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] .", "Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] . However, the extreme mutation and high replication rates of HCV, together with the immune system pressure, lead to a remarkable genetic variability that can compromise the high response rates to DAAs due to the preexistence of resistanceassociated substitutions (RASs) [5, 6] . Each drug or class of DAA is characterized by specific resistance profiles.", "Each drug or class of DAA is characterized by specific resistance profiles. The likelihood that a DAA will select for and allow outgrowth of viral populations carrying RASs depends on the DAA's genetic barrier to resistance (the number and type of mutations needed to generate an amino acid substitution that confers resistance), the viral fitness (replicative capacity) of the resistant variant, and viral genotypes and subtypes [7, 8] . The prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] .", "The prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] . However, apart from Brazil and Argentina, this issue has not been fully addressed in other South American countries yet [9, [17] [18] [19] . The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] .", "The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] . In this study, we explored the presence of resistance variants to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients chronically infected with hepatitis C. Here, we aimed to contribute to the knowledge of the circulation of HCV resistant variants in the South American region. Samples.", "Samples. Serum samples were obtained from 31 patients with serological markers for HCV, which were recruited between 2015 and 2017 at the Gastroenterology Clinic from Hospital de Clínicas, Montevideo, Uruguay. HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA).", "HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA). Patients selected for this study were both chronically infected with HCV genotype 1 and DAA treatment-naïve at the time of blood extraction. Written informed consent was obtained from all patients.", "Written informed consent was obtained from all patients. The studies have been performed according to the World Medical Association Declaration of Helsinki and approved by the appropriate institutional board (Hospital de Clínicas ethical committee). 2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification.", "2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification. Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol.", "Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol. The viral RNA was heated at 65°C for 5 min and used as a template for a reverse transcription reaction. The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA).", "The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA). The reverse transcription was performed at 42°C for 50 min, and then the reverse transcriptase enzyme was inactivated at 70°C for 15 min. PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously described [10] .", "PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously described [10] . Amplicons were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Science, Buckinghamshire, UK) according to the manufacturer's protocol. 2.3. NS5A and NS5B Sequencing.", "2.3. NS5A and NS5B Sequencing. The purified product was then sequenced using the same sets of primers used for PCR amplification. Bidirectional Sanger sequencing was performed by Macrogen Korea (\n\n2.4. NS5A and NS5B Genotype Determination.", "NS5A and NS5B Genotype Determination. HCV NS5A and NS5B consensus sequences obtained from Uruguayan patients were aligned with sequences from HCV representing all genotypes and main subtypes isolated in different geographic regions of the world. These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] .", "These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] . For strains included in these studies, see Supplementary Material Table S1 . Sequences were aligned using the CLUSTAL W software [23] .", "Sequences were aligned using the CLUSTAL W software [23] . Once aligned, the best evolutionary model that described our sequence data was assessed using ModelGenerator program [24] . Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] .", "Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] . For NS5A, 953 nucleotides (positions 6367 to 7319, relative to HCV 1a reference strain, H77 NC_004102) were included in the phylogenetic analysis, whereas for NS5B, only 361 nucleotides corresponding to the Okamoto region (positions 8265 to 8625, relative to strain H77 NC_004102) were included. As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates).", "As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates). For NS5A 1a Uruguayan sequences (n = 20), a second alignment and maximum likelihood phylogenetic tree was generated in order to analyze HCV evolutionary relationships between Uruguayan, Brazilian, and worldwide strains. For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2.", "For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2. 2.5. NS5A and NS5B Sequence Analysis.", "2.5. NS5A and NS5B Sequence Analysis. In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide.", "In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide. For this purpose, NS5A gene sequences corresponding to subtypes 1a (n = 160) and 1b (n = 88) were retrieved from Los Alamos HCV sequence database and from the NIAID ViPR [21, 22] . Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively.", "Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively. Using Seqman program, implemented in DNAStar 5.01 package (DNASTAR, Madison, USA), a world consensus nucleotide sequences were generated for each gene and subtype. Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated.", "Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated. The amino acid sequences obtained were compared in order to explore the presence of RASs as well as the presence of polymorphisms at a RAS position (RAPs) in Uruguayan HCV strains. RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] .", "RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] . To study the genetic variability of NS5A and NS5B regions of HCV strains circulating in Uruguayan patients, sequences of these regions (accession numbers MH070029-MH070090) were aligned with corresponding sequences from 59 HCV strains isolated elsewhere, representing all genotypes and main subtypes (for strains included in these analyses, see Supplementary Material Table S1 ). Therefore, maximum likelihood phylogenetic trees were constructed.", "Therefore, maximum likelihood phylogenetic trees were constructed. The results of these studies are shown in Figure 1 All strains in the phylogenies were assigned according to their genotype, and each cluster was supported by very high bootstrap values for both analyzed regions. Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b.", "Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b. The results of NS5A (Figure 1 (a)) and NS5B (Figure 1 \n\nGenotype 1b phylogenetic analyses were concordant for both genomic regions in all 31 sequences, suggesting no recombination events between these regions. To further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ).", "To further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ). As was previously described, two distinct 1a clades (clades 1 and 2) were observed. Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] .", "Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] . Whereas NS5A Uruguayan strains (in red) did not cluster in a particular clade, rather, they grouped dispersedly within all major world clades. With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence).", "With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence). AA substitutions at positions previously found to be potentially associated with resistance to NS5A inhibitors, as well as polymorphisms at a RAS position, were identified. These results are summarized in Table 1 .", "These results are summarized in Table 1 . RASs to NS5A inhibitors (L31M and L31V) were identified in 2 strains out of 25 (8%) fully sequenced samples. RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q.", "RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q. Although these substitutions were not reported as resistant, some changes at these positions were previously described as RASs in subtype 1a, namely H58D and K24R [27, 28] . Finally, substitution E62D was found in one subtype 1a strain.", "Finally, substitution E62D was found in one subtype 1a strain. This change is considered as a secondary substitution because, although it does not confer resistance by itself, when combined with a known RAS it does. In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] .", "In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] . In addition, several polymorphisms that have not been previously reported to be associated with a resistant phenotype were also detected (see Supplementary Material Table S3 ). In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences.", "In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences. Almost full-length AA sequences were obtained in 26 out of 31 analyzed strains. 23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein.", "23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein. This issue limited our studies, since many of the described RASs are observed as of residue 553. Importantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%).", "Importantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%). C451R was found in two isolates while A421V was found in only one. In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed.", "In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed. Finally, we found two RAPs: A421V (in 2 subtype 1b strains) and A553G (in 1 subtype 1a strain). Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] .", "Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] . In position 553, the substitution reported as resistant was A553T [8] . As was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ).", "As was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ). The advent of DAAs therapies constitutes one of the major breakthroughs in HCV infected patients management. However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems.", "However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems. The main limiting factors for worldwide access to DAAs in our region concern the high cost, the inadequate management of public healthcare systems, the limited access of low-income or uninsured populations to healthcare providers, and the lack of accurate epidemiological information [20, [30] [31] [32] . In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases.", "In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases. Despite the high rates of viral response achieved with DAA-based treatments, still 1 to10% of the patients fails to eliminate infection, and in these cases, baseline and emergent resistance variants turn out to be key factors contributing to treatment failure [5, 17, 33] . Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating.", "Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating. These facts could compromise the effectiveness of these new therapies in our country. We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] .", "We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] . The present study describes the prevalence of baseline NS5A and NS5B RASs in HCV genotype 1 infected DAA-naïve patients in a Uruguayan cohort. The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] .", "The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] . However, NS5A sequences from South America are poorly analyzed yet [9, 44] . Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] .", "Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] . Importantly, the prevalence and type of baseline NS5A RASs varies slightly by geographic regions. For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] .", "For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] . For this reason, we believe that there is a need to contribute data from our region, for which we still do not have enough information, apart from Brazil [9, 44] . The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ).", "The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ). L31M substitution confers resistance to daclatasvir (DCV), ledipasvir (LDV), and elbasvir (EBV) in both 1a and 1b subtypes [5, 6, 8, 28, 47, 48] , whereas substitution L31V does it to DCV in subtypes 1a and 1b, to LDV in subtype 1b, and to EBV in subtype 1a [5, 6, 28] . Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] .", "Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] . The substitutions H58P and K24Q found in two patients are considered as resistance-associated polymorphisms (RAPs). The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] .", "The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] . The substitution H58P was found as a baseline RAP in relapsers to LDV (HARVONI prescription, media/files/pdfs/medicines/liver-disease/harvoni/harvoni_pi. pdf?la=en).", "pdf?la=en). However, it is sometimes regarded as a RAS [10, 51] , despite conferring only 1.2 fold change in resistance in in vitro studies using the 1a replicon system [39] . We did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] .", "We did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] . The amino acid substitution E62H was found in one Uruguayan patient. Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] .", "Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] . The presence of baseline NS5A RASs impacts treatment outcome in some patient groups by affecting SVR rates. The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use.", "The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use. Regarding NS5B gene, global analysis (with the exception of South America [17, 19] ) revealed that NS5B DAA resistance substitutions are infrequent [14] . Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%).", "Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%). Substitutions found in this work were A421V and S556G associated in subtype 1a with resistance to BCV and dasabuvir (DSV), respectively [8, 28, 29, 54, 55] , and Q556R associated with resistance to DSV both in genotype 1a and 1b [12, 28] . Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV.", "Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV. In these cases, it appeared in combination with G558R (Trial Coral I-Cohort 2: RAPs in positions 421 and 553 (A421V in two subtype 1b isolates and A553G in one subtype 1b isolate) were also found. Although A421V has been associated with resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] .", "Although A421V has been associated with resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] . In position 553, the substitutions reported as resistant are A553T in subtype 1a [8] and A553V in subtype 1b [54] , conferring resistance to DSV. In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ).", "In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ). Nevertheless, substitution A421V was found in Brazil [17] , Argentina [19] , and Uruguay. The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] .", "The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] . Our findings further confirm and complement previous studies which evidenced a low prevalence of this substitution in vivo, probably due to its low replicative fitness [14, 18, 57] . Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] .", "Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] . These results show both diversity in the baseline polymorphisms found in different Latin American countries and in the evolutionary relationships of Uruguayan isolates ( Figure 2 ). This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host.", "This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host. It is worth mentioning that we live in a vast continent inhabited by populations with different genotypic characteristics that might, depending on the situation, require different approaches to treatment. Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] .", "Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] . Altogether, we believe that it could be important to carry out studies throughout the South American region in order to establish the prevalence of RASs in NS5A and NS5B in different countries. In fact, this will aid in understanding that not every treatment regimen might be adequate for every patient and country.", "In fact, this will aid in understanding that not every treatment regimen might be adequate for every patient and country. The data we presented here might guide not only physicians in making therapeutic decisions but also public health authorities in approving more diverse treatment combinations. These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population.", "These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population. To our knowledge, the present study revealed for the first time the presence of RASs in the NS5A and NS5B regions of HCV genotype 1 Uruguayan strains from patients who have not been previously treated with DAAs and is one of the few South American countries to report on this matter. It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure.", "It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure. However, individualized DAA therapy based on baseline resistance analysis may be beneficial for optimizing treatment efficacy in patients with HCV genotype 1 infection and risk factors for treatment failure. Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions.", "Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions. The data used to support the findings of this study are included within the article. The authors declare that they have no conflicts of interest. Fabián Aldunate and Natalia Echeverría contributed equally to this work.", "Fabián Aldunate and Natalia Echeverría contributed equally to this work. Supplementary Material Table S1 : hepatitis C Virus NS5A and NS5B sequences used as representatives of each genotype to perform the phylogenetic analysis. Their corresponding genotype, country of isolation, and GenBank accession number are indicated.", "Their corresponding genotype, country of isolation, and GenBank accession number are indicated. Supplementary Material Table S2 : hepatitis C Virus NS5A subtype 1a sequences used to reveal evolutionary relationships between Uruguayan strains and others isolated elsewhere. Their corresponding country of isolation and GenBank accession number are indicated.", "Their corresponding country of isolation and GenBank accession number are indicated. Supplementary Material Table S3 : amino acid substitutions in NS5A protein not previously associated with resistance to NS5A inhibitors. Supplementary Material Table S4 : amino acid substitutions in NS5B protein not previously associated with resistance to polymerase inhibitors. (Supplementary Materials)" ]

[ "Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction.", "Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries.", "The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil.", "Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity. Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs).", "Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs). These antivirals have significantly increased response rates (up to 98%) and greatly reduced treatment duration [1] . Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] .", "Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] . However, the extreme mutation and high replication rates of HCV, together with the immune system pressure, lead to a remarkable genetic variability that can compromise the high response rates to DAAs due to the preexistence of resistanceassociated substitutions (RASs) [5, 6] . Each drug or class of DAA is characterized by specific resistance profiles.", "Each drug or class of DAA is characterized by specific resistance profiles. The likelihood that a DAA will select for and allow outgrowth of viral populations carrying RASs depends on the DAA's genetic barrier to resistance (the number and type of mutations needed to generate an amino acid substitution that confers resistance), the viral fitness (replicative capacity) of the resistant variant, and viral genotypes and subtypes [7, 8] . The prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] .", "The prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] . However, apart from Brazil and Argentina, this issue has not been fully addressed in other South American countries yet [9, [17] [18] [19] . The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] .", "The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] . In this study, we explored the presence of resistance variants to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients chronically infected with hepatitis C. Here, we aimed to contribute to the knowledge of the circulation of HCV resistant variants in the South American region. Samples.", "Samples. Serum samples were obtained from 31 patients with serological markers for HCV, which were recruited between 2015 and 2017 at the Gastroenterology Clinic from Hospital de Clínicas, Montevideo, Uruguay. HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA).", "HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA). Patients selected for this study were both chronically infected with HCV genotype 1 and DAA treatment-naïve at the time of blood extraction. Written informed consent was obtained from all patients.", "Written informed consent was obtained from all patients. The studies have been performed according to the World Medical Association Declaration of Helsinki and approved by the appropriate institutional board (Hospital de Clínicas ethical committee). 2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification.", "2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification. Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol.", "Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol. The viral RNA was heated at 65°C for 5 min and used as a template for a reverse transcription reaction. The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA).", "The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA). The reverse transcription was performed at 42°C for 50 min, and then the reverse transcriptase enzyme was inactivated at 70°C for 15 min. PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously described [10] .", "PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously described [10] . Amplicons were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Science, Buckinghamshire, UK) according to the manufacturer's protocol. 2.3. NS5A and NS5B Sequencing.", "2.3. NS5A and NS5B Sequencing. The purified product was then sequenced using the same sets of primers used for PCR amplification. Bidirectional Sanger sequencing was performed by Macrogen Korea (\n\n2.4. NS5A and NS5B Genotype Determination.", "NS5A and NS5B Genotype Determination. HCV NS5A and NS5B consensus sequences obtained from Uruguayan patients were aligned with sequences from HCV representing all genotypes and main subtypes isolated in different geographic regions of the world. These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] .", "These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] . For strains included in these studies, see Supplementary Material Table S1 . Sequences were aligned using the CLUSTAL W software [23] .", "Sequences were aligned using the CLUSTAL W software [23] . Once aligned, the best evolutionary model that described our sequence data was assessed using ModelGenerator program [24] . Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] .", "Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] . For NS5A, 953 nucleotides (positions 6367 to 7319, relative to HCV 1a reference strain, H77 NC_004102) were included in the phylogenetic analysis, whereas for NS5B, only 361 nucleotides corresponding to the Okamoto region (positions 8265 to 8625, relative to strain H77 NC_004102) were included. As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates).", "As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates). For NS5A 1a Uruguayan sequences (n = 20), a second alignment and maximum likelihood phylogenetic tree was generated in order to analyze HCV evolutionary relationships between Uruguayan, Brazilian, and worldwide strains. For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2.", "For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2. 2.5. NS5A and NS5B Sequence Analysis.", "2.5. NS5A and NS5B Sequence Analysis. In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide.", "In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide. For this purpose, NS5A gene sequences corresponding to subtypes 1a (n = 160) and 1b (n = 88) were retrieved from Los Alamos HCV sequence database and from the NIAID ViPR [21, 22] . Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively.", "Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively. Using Seqman program, implemented in DNAStar 5.01 package (DNASTAR, Madison, USA), a world consensus nucleotide sequences were generated for each gene and subtype. Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated.", "Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated. The amino acid sequences obtained were compared in order to explore the presence of RASs as well as the presence of polymorphisms at a RAS position (RAPs) in Uruguayan HCV strains. RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] .", "RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] . To study the genetic variability of NS5A and NS5B regions of HCV strains circulating in Uruguayan patients, sequences of these regions (accession numbers MH070029-MH070090) were aligned with corresponding sequences from 59 HCV strains isolated elsewhere, representing all genotypes and main subtypes (for strains included in these analyses, see Supplementary Material Table S1 ). Therefore, maximum likelihood phylogenetic trees were constructed.", "Therefore, maximum likelihood phylogenetic trees were constructed. The results of these studies are shown in Figure 1 All strains in the phylogenies were assigned according to their genotype, and each cluster was supported by very high bootstrap values for both analyzed regions. Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b.", "Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b. The results of NS5A (Figure 1 (a)) and NS5B (Figure 1 \n\nGenotype 1b phylogenetic analyses were concordant for both genomic regions in all 31 sequences, suggesting no recombination events between these regions. To further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ).", "To further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ). As was previously described, two distinct 1a clades (clades 1 and 2) were observed. Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] .", "Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] . Whereas NS5A Uruguayan strains (in red) did not cluster in a particular clade, rather, they grouped dispersedly within all major world clades. With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence).", "With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence). AA substitutions at positions previously found to be potentially associated with resistance to NS5A inhibitors, as well as polymorphisms at a RAS position, were identified. These results are summarized in Table 1 .", "These results are summarized in Table 1 . RASs to NS5A inhibitors (L31M and L31V) were identified in 2 strains out of 25 (8%) fully sequenced samples. RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q.", "RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q. Although these substitutions were not reported as resistant, some changes at these positions were previously described as RASs in subtype 1a, namely H58D and K24R [27, 28] . Finally, substitution E62D was found in one subtype 1a strain.", "Finally, substitution E62D was found in one subtype 1a strain. This change is considered as a secondary substitution because, although it does not confer resistance by itself, when combined with a known RAS it does. In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] .", "In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] . In addition, several polymorphisms that have not been previously reported to be associated with a resistant phenotype were also detected (see Supplementary Material Table S3 ). In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences.", "In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences. Almost full-length AA sequences were obtained in 26 out of 31 analyzed strains. 23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein.", "23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein. This issue limited our studies, since many of the described RASs are observed as of residue 553. Importantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%).", "Importantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%). C451R was found in two isolates while A421V was found in only one. In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed.", "In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed. Finally, we found two RAPs: A421V (in 2 subtype 1b strains) and A553G (in 1 subtype 1a strain). Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] .", "Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] . In position 553, the substitution reported as resistant was A553T [8] . As was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ).", "As was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ). The advent of DAAs therapies constitutes one of the major breakthroughs in HCV infected patients management. However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems.", "However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems. The main limiting factors for worldwide access to DAAs in our region concern the high cost, the inadequate management of public healthcare systems, the limited access of low-income or uninsured populations to healthcare providers, and the lack of accurate epidemiological information [20, [30] [31] [32] . In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases.", "In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases. Despite the high rates of viral response achieved with DAA-based treatments, still 1 to10% of the patients fails to eliminate infection, and in these cases, baseline and emergent resistance variants turn out to be key factors contributing to treatment failure [5, 17, 33] . Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating.", "Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating. These facts could compromise the effectiveness of these new therapies in our country. We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] .", "We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] . The present study describes the prevalence of baseline NS5A and NS5B RASs in HCV genotype 1 infected DAA-naïve patients in a Uruguayan cohort. The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] .", "The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] . However, NS5A sequences from South America are poorly analyzed yet [9, 44] . Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] .", "Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] . Importantly, the prevalence and type of baseline NS5A RASs varies slightly by geographic regions. For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] .", "For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] . For this reason, we believe that there is a need to contribute data from our region, for which we still do not have enough information, apart from Brazil [9, 44] . The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ).", "The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ). L31M substitution confers resistance to daclatasvir (DCV), ledipasvir (LDV), and elbasvir (EBV) in both 1a and 1b subtypes [5, 6, 8, 28, 47, 48] , whereas substitution L31V does it to DCV in subtypes 1a and 1b, to LDV in subtype 1b, and to EBV in subtype 1a [5, 6, 28] . Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] .", "Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] . The substitutions H58P and K24Q found in two patients are considered as resistance-associated polymorphisms (RAPs). The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] .", "The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] . The substitution H58P was found as a baseline RAP in relapsers to LDV (HARVONI prescription, media/files/pdfs/medicines/liver-disease/harvoni/harvoni_pi. pdf?la=en).", "pdf?la=en). However, it is sometimes regarded as a RAS [10, 51] , despite conferring only 1.2 fold change in resistance in in vitro studies using the 1a replicon system [39] . We did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] .", "We did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] . The amino acid substitution E62H was found in one Uruguayan patient. Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] .", "Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] . The presence of baseline NS5A RASs impacts treatment outcome in some patient groups by affecting SVR rates. The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use.", "The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use. Regarding NS5B gene, global analysis (with the exception of South America [17, 19] ) revealed that NS5B DAA resistance substitutions are infrequent [14] . Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%).", "Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%). Substitutions found in this work were A421V and S556G associated in subtype 1a with resistance to BCV and dasabuvir (DSV), respectively [8, 28, 29, 54, 55] , and Q556R associated with resistance to DSV both in genotype 1a and 1b [12, 28] . Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV.", "Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV. In these cases, it appeared in combination with G558R (Trial Coral I-Cohort 2: RAPs in positions 421 and 553 (A421V in two subtype 1b isolates and A553G in one subtype 1b isolate) were also found. Although A421V has been associated with resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] .", "Although A421V has been associated with resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] . In position 553, the substitutions reported as resistant are A553T in subtype 1a [8] and A553V in subtype 1b [54] , conferring resistance to DSV. In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ).", "In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ). Nevertheless, substitution A421V was found in Brazil [17] , Argentina [19] , and Uruguay. The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] .", "The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] . Our findings further confirm and complement previous studies which evidenced a low prevalence of this substitution in vivo, probably due to its low replicative fitness [14, 18, 57] . Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] .", "Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] . These results show both diversity in the baseline polymorphisms found in different Latin American countries and in the evolutionary relationships of Uruguayan isolates ( Figure 2 ). This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host.", "This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host. It is worth mentioning that we live in a vast continent inhabited by populations with different genotypic characteristics that might, depending on the situation, require different approaches to treatment. Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] .", "Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] . Altogether, we believe that it could be important to carry out studies throughout the South American region in order to establish the prevalence of RASs in NS5A and NS5B in different countries. In fact, this will aid in understanding that not every treatment regimen might be adequate for every patient and country.", "In fact, this will aid in understanding that not every treatment regimen might be adequate for every patient and country. The data we presented here might guide not only physicians in making therapeutic decisions but also public health authorities in approving more diverse treatment combinations. These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population.", "These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population. To our knowledge, the present study revealed for the first time the presence of RASs in the NS5A and NS5B regions of HCV genotype 1 Uruguayan strains from patients who have not been previously treated with DAAs and is one of the few South American countries to report on this matter. It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure.", "It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure. However, individualized DAA therapy based on baseline resistance analysis may be beneficial for optimizing treatment efficacy in patients with HCV genotype 1 infection and risk factors for treatment failure. Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions.", "Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions. The data used to support the findings of this study are included within the article. The authors declare that they have no conflicts of interest. Fabián Aldunate and Natalia Echeverría contributed equally to this work.", "Fabián Aldunate and Natalia Echeverría contributed equally to this work. Supplementary Material Table S1 : hepatitis C Virus NS5A and NS5B sequences used as representatives of each genotype to perform the phylogenetic analysis. Their corresponding genotype, country of isolation, and GenBank accession number are indicated.", "Their corresponding genotype, country of isolation, and GenBank accession number are indicated. Supplementary Material Table S2 : hepatitis C Virus NS5A subtype 1a sequences used to reveal evolutionary relationships between Uruguayan strains and others isolated elsewhere. Their corresponding country of isolation and GenBank accession number are indicated.", "Their corresponding country of isolation and GenBank accession number are indicated. Supplementary Material Table S3 : amino acid substitutions in NS5A protein not previously associated with resistance to NS5A inhibitors. Supplementary Material Table S4 : amino acid substitutions in NS5B protein not previously associated with resistance to polymerase inhibitors. (Supplementary Materials)" ]

[ "Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction.", "Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries.", "The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil.", "Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity. Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs).", "Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs). These antivirals have significantly increased response rates (up to 98%) and greatly reduced treatment duration [1] . Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] .", "Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] . However, the extreme mutation and high replication rates of HCV, together with the immune system pressure, lead to a remarkable genetic variability that can compromise the high response rates to DAAs due to the preexistence of resistanceassociated substitutions (RASs) [5, 6] . Each drug or class of DAA is characterized by specific resistance profiles.", "Each drug or class of DAA is characterized by specific resistance profiles. The likelihood that a DAA will select for and allow outgrowth of viral populations carrying RASs depends on the DAA's genetic barrier to resistance (the number and type of mutations needed to generate an amino acid substitution that confers resistance), the viral fitness (replicative capacity) of the resistant variant, and viral genotypes and subtypes [7, 8] . The prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] .", "The prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] . However, apart from Brazil and Argentina, this issue has not been fully addressed in other South American countries yet [9, [17] [18] [19] . The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] .", "The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] . In this study, we explored the presence of resistance variants to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients chronically infected with hepatitis C. Here, we aimed to contribute to the knowledge of the circulation of HCV resistant variants in the South American region. Samples.", "Samples. Serum samples were obtained from 31 patients with serological markers for HCV, which were recruited between 2015 and 2017 at the Gastroenterology Clinic from Hospital de Clínicas, Montevideo, Uruguay. HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA).", "HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA). Patients selected for this study were both chronically infected with HCV genotype 1 and DAA treatment-naïve at the time of blood extraction. Written informed consent was obtained from all patients.", "Written informed consent was obtained from all patients. The studies have been performed according to the World Medical Association Declaration of Helsinki and approved by the appropriate institutional board (Hospital de Clínicas ethical committee). 2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification.", "2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification. Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol.", "Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol. The viral RNA was heated at 65°C for 5 min and used as a template for a reverse transcription reaction. The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA).", "The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA). The reverse transcription was performed at 42°C for 50 min, and then the reverse transcriptase enzyme was inactivated at 70°C for 15 min. PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously described [10] .", "PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously described [10] . Amplicons were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Science, Buckinghamshire, UK) according to the manufacturer's protocol. 2.3. NS5A and NS5B Sequencing.", "2.3. NS5A and NS5B Sequencing. The purified product was then sequenced using the same sets of primers used for PCR amplification. Bidirectional Sanger sequencing was performed by Macrogen Korea (\n\n2.4. NS5A and NS5B Genotype Determination.", "NS5A and NS5B Genotype Determination. HCV NS5A and NS5B consensus sequences obtained from Uruguayan patients were aligned with sequences from HCV representing all genotypes and main subtypes isolated in different geographic regions of the world. These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] .", "These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] . For strains included in these studies, see Supplementary Material Table S1 . Sequences were aligned using the CLUSTAL W software [23] .", "Sequences were aligned using the CLUSTAL W software [23] . Once aligned, the best evolutionary model that described our sequence data was assessed using ModelGenerator program [24] . Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] .", "Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] . For NS5A, 953 nucleotides (positions 6367 to 7319, relative to HCV 1a reference strain, H77 NC_004102) were included in the phylogenetic analysis, whereas for NS5B, only 361 nucleotides corresponding to the Okamoto region (positions 8265 to 8625, relative to strain H77 NC_004102) were included. As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates).", "As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates). For NS5A 1a Uruguayan sequences (n = 20), a second alignment and maximum likelihood phylogenetic tree was generated in order to analyze HCV evolutionary relationships between Uruguayan, Brazilian, and worldwide strains. For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2.", "For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2. 2.5. NS5A and NS5B Sequence Analysis.", "2.5. NS5A and NS5B Sequence Analysis. In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide.", "In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide. For this purpose, NS5A gene sequences corresponding to subtypes 1a (n = 160) and 1b (n = 88) were retrieved from Los Alamos HCV sequence database and from the NIAID ViPR [21, 22] . Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively.", "Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively. Using Seqman program, implemented in DNAStar 5.01 package (DNASTAR, Madison, USA), a world consensus nucleotide sequences were generated for each gene and subtype. Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated.", "Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated. The amino acid sequences obtained were compared in order to explore the presence of RASs as well as the presence of polymorphisms at a RAS position (RAPs) in Uruguayan HCV strains. RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] .", "RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] . To study the genetic variability of NS5A and NS5B regions of HCV strains circulating in Uruguayan patients, sequences of these regions (accession numbers MH070029-MH070090) were aligned with corresponding sequences from 59 HCV strains isolated elsewhere, representing all genotypes and main subtypes (for strains included in these analyses, see Supplementary Material Table S1 ). Therefore, maximum likelihood phylogenetic trees were constructed.", "Therefore, maximum likelihood phylogenetic trees were constructed. The results of these studies are shown in Figure 1 All strains in the phylogenies were assigned according to their genotype, and each cluster was supported by very high bootstrap values for both analyzed regions. Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b.", "Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b. The results of NS5A (Figure 1 (a)) and NS5B (Figure 1 \n\nGenotype 1b phylogenetic analyses were concordant for both genomic regions in all 31 sequences, suggesting no recombination events between these regions. To further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ).", "To further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ). As was previously described, two distinct 1a clades (clades 1 and 2) were observed. Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] .", "Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] . Whereas NS5A Uruguayan strains (in red) did not cluster in a particular clade, rather, they grouped dispersedly within all major world clades. With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence).", "With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence). AA substitutions at positions previously found to be potentially associated with resistance to NS5A inhibitors, as well as polymorphisms at a RAS position, were identified. These results are summarized in Table 1 .", "These results are summarized in Table 1 . RASs to NS5A inhibitors (L31M and L31V) were identified in 2 strains out of 25 (8%) fully sequenced samples. RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q.", "RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q. Although these substitutions were not reported as resistant, some changes at these positions were previously described as RASs in subtype 1a, namely H58D and K24R [27, 28] . Finally, substitution E62D was found in one subtype 1a strain.", "Finally, substitution E62D was found in one subtype 1a strain. This change is considered as a secondary substitution because, although it does not confer resistance by itself, when combined with a known RAS it does. In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] .", "In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] . In addition, several polymorphisms that have not been previously reported to be associated with a resistant phenotype were also detected (see Supplementary Material Table S3 ). In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences.", "In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences. Almost full-length AA sequences were obtained in 26 out of 31 analyzed strains. 23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein.", "23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein. This issue limited our studies, since many of the described RASs are observed as of residue 553. Importantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%).", "Importantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%). C451R was found in two isolates while A421V was found in only one. In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed.", "In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed. Finally, we found two RAPs: A421V (in 2 subtype 1b strains) and A553G (in 1 subtype 1a strain). Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] .", "Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] . In position 553, the substitution reported as resistant was A553T [8] . As was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ).", "As was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ). The advent of DAAs therapies constitutes one of the major breakthroughs in HCV infected patients management. However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems.", "However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems. The main limiting factors for worldwide access to DAAs in our region concern the high cost, the inadequate management of public healthcare systems, the limited access of low-income or uninsured populations to healthcare providers, and the lack of accurate epidemiological information [20, [30] [31] [32] . In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases.", "In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases. Despite the high rates of viral response achieved with DAA-based treatments, still 1 to10% of the patients fails to eliminate infection, and in these cases, baseline and emergent resistance variants turn out to be key factors contributing to treatment failure [5, 17, 33] . Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating.", "Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating. These facts could compromise the effectiveness of these new therapies in our country. We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] .", "We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] . The present study describes the prevalence of baseline NS5A and NS5B RASs in HCV genotype 1 infected DAA-naïve patients in a Uruguayan cohort. The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] .", "The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] . However, NS5A sequences from South America are poorly analyzed yet [9, 44] . Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] .", "Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] . Importantly, the prevalence and type of baseline NS5A RASs varies slightly by geographic regions. For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] .", "For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] . For this reason, we believe that there is a need to contribute data from our region, for which we still do not have enough information, apart from Brazil [9, 44] . The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ).", "The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ). L31M substitution confers resistance to daclatasvir (DCV), ledipasvir (LDV), and elbasvir (EBV) in both 1a and 1b subtypes [5, 6, 8, 28, 47, 48] , whereas substitution L31V does it to DCV in subtypes 1a and 1b, to LDV in subtype 1b, and to EBV in subtype 1a [5, 6, 28] . Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] .", "Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] . The substitutions H58P and K24Q found in two patients are considered as resistance-associated polymorphisms (RAPs). The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] .", "The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] . The substitution H58P was found as a baseline RAP in relapsers to LDV (HARVONI prescription, media/files/pdfs/medicines/liver-disease/harvoni/harvoni_pi. pdf?la=en).", "pdf?la=en). However, it is sometimes regarded as a RAS [10, 51] , despite conferring only 1.2 fold change in resistance in in vitro studies using the 1a replicon system [39] . We did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] .", "We did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] . The amino acid substitution E62H was found in one Uruguayan patient. Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] .", "Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] . The presence of baseline NS5A RASs impacts treatment outcome in some patient groups by affecting SVR rates. The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use.", "The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use. Regarding NS5B gene, global analysis (with the exception of South America [17, 19] ) revealed that NS5B DAA resistance substitutions are infrequent [14] . Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%).", "Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%). Substitutions found in this work were A421V and S556G associated in subtype 1a with resistance to BCV and dasabuvir (DSV), respectively [8, 28, 29, 54, 55] , and Q556R associated with resistance to DSV both in genotype 1a and 1b [12, 28] . Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV.", "Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV. In these cases, it appeared in combination with G558R (Trial Coral I-Cohort 2: RAPs in positions 421 and 553 (A421V in two subtype 1b isolates and A553G in one subtype 1b isolate) were also found. Although A421V has been associated with resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] .", "Although A421V has been associated with resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] . In position 553, the substitutions reported as resistant are A553T in subtype 1a [8] and A553V in subtype 1b [54] , conferring resistance to DSV. In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ).", "In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ). Nevertheless, substitution A421V was found in Brazil [17] , Argentina [19] , and Uruguay. The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] .", "The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] . Our findings further confirm and complement previous studies which evidenced a low prevalence of this substitution in vivo, probably due to its low replicative fitness [14, 18, 57] . Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] .", "Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] . These results show both diversity in the baseline polymorphisms found in different Latin American countries and in the evolutionary relationships of Uruguayan isolates ( Figure 2 ). This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host.", "This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host. It is worth mentioning that we live in a vast continent inhabited by populations with different genotypic characteristics that might, depending on the situation, require different approaches to treatment. Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] .", "Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] . Altogether, we believe that it could be important to carry out studies throughout the South American region in order to establish the prevalence of RASs in NS5A and NS5B in different countries. In fact, this will aid in understanding that not every treatment regimen might be adequate for every patient and country.", "In fact, this will aid in understanding that not every treatment regimen might be adequate for every patient and country. The data we presented here might guide not only physicians in making therapeutic decisions but also public health authorities in approving more diverse treatment combinations. These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population.", "These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population. To our knowledge, the present study revealed for the first time the presence of RASs in the NS5A and NS5B regions of HCV genotype 1 Uruguayan strains from patients who have not been previously treated with DAAs and is one of the few South American countries to report on this matter. It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure.", "It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure. However, individualized DAA therapy based on baseline resistance analysis may be beneficial for optimizing treatment efficacy in patients with HCV genotype 1 infection and risk factors for treatment failure. Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions.", "Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions. The data used to support the findings of this study are included within the article. The authors declare that they have no conflicts of interest. Fabián Aldunate and Natalia Echeverría contributed equally to this work.", "Fabián Aldunate and Natalia Echeverría contributed equally to this work. Supplementary Material Table S1 : hepatitis C Virus NS5A and NS5B sequences used as representatives of each genotype to perform the phylogenetic analysis. Their corresponding genotype, country of isolation, and GenBank accession number are indicated.", "Their corresponding genotype, country of isolation, and GenBank accession number are indicated. Supplementary Material Table S2 : hepatitis C Virus NS5A subtype 1a sequences used to reveal evolutionary relationships between Uruguayan strains and others isolated elsewhere. Their corresponding country of isolation and GenBank accession number are indicated.", "Their corresponding country of isolation and GenBank accession number are indicated. Supplementary Material Table S3 : amino acid substitutions in NS5A protein not previously associated with resistance to NS5A inhibitors. Supplementary Material Table S4 : amino acid substitutions in NS5B protein not previously associated with resistance to polymerase inhibitors. (Supplementary Materials)" ]

[ "Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction.", "Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries.", "The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil.", "Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity. Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs).", "Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs). These antivirals have significantly increased response rates (up to 98%) and greatly reduced treatment duration [1] . Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] .", "Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] . However, the extreme mutation and high replication rates of HCV, together with the immune system pressure, lead to a remarkable genetic variability that can compromise the high response rates to DAAs due to the preexistence of resistanceassociated substitutions (RASs) [5, 6] . Each drug or class of DAA is characterized by specific resistance profiles.", "Each drug or class of DAA is characterized by specific resistance profiles. The likelihood that a DAA will select for and allow outgrowth of viral populations carrying RASs depends on the DAA's genetic barrier to resistance (the number and type of mutations needed to generate an amino acid substitution that confers resistance), the viral fitness (replicative capacity) of the resistant variant, and viral genotypes and subtypes [7, 8] . The prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] .", "The prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] . However, apart from Brazil and Argentina, this issue has not been fully addressed in other South American countries yet [9, [17] [18] [19] . The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] .", "The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] . In this study, we explored the presence of resistance variants to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients chronically infected with hepatitis C. Here, we aimed to contribute to the knowledge of the circulation of HCV resistant variants in the South American region. Samples.", "Samples. Serum samples were obtained from 31 patients with serological markers for HCV, which were recruited between 2015 and 2017 at the Gastroenterology Clinic from Hospital de Clínicas, Montevideo, Uruguay. HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA).", "HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA). Patients selected for this study were both chronically infected with HCV genotype 1 and DAA treatment-naïve at the time of blood extraction. Written informed consent was obtained from all patients.", "Written informed consent was obtained from all patients. The studies have been performed according to the World Medical Association Declaration of Helsinki and approved by the appropriate institutional board (Hospital de Clínicas ethical committee). 2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification.", "2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification. Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol.", "Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol. The viral RNA was heated at 65°C for 5 min and used as a template for a reverse transcription reaction. The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA).", "The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA). The reverse transcription was performed at 42°C for 50 min, and then the reverse transcriptase enzyme was inactivated at 70°C for 15 min. PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously described [10] .", "PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously described [10] . Amplicons were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Science, Buckinghamshire, UK) according to the manufacturer's protocol. 2.3. NS5A and NS5B Sequencing.", "2.3. NS5A and NS5B Sequencing. The purified product was then sequenced using the same sets of primers used for PCR amplification. Bidirectional Sanger sequencing was performed by Macrogen Korea (\n\n2.4. NS5A and NS5B Genotype Determination.", "NS5A and NS5B Genotype Determination. HCV NS5A and NS5B consensus sequences obtained from Uruguayan patients were aligned with sequences from HCV representing all genotypes and main subtypes isolated in different geographic regions of the world. These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] .", "These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] . For strains included in these studies, see Supplementary Material Table S1 . Sequences were aligned using the CLUSTAL W software [23] .", "Sequences were aligned using the CLUSTAL W software [23] . Once aligned, the best evolutionary model that described our sequence data was assessed using ModelGenerator program [24] . Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] .", "Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] . For NS5A, 953 nucleotides (positions 6367 to 7319, relative to HCV 1a reference strain, H77 NC_004102) were included in the phylogenetic analysis, whereas for NS5B, only 361 nucleotides corresponding to the Okamoto region (positions 8265 to 8625, relative to strain H77 NC_004102) were included. As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates).", "As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates). For NS5A 1a Uruguayan sequences (n = 20), a second alignment and maximum likelihood phylogenetic tree was generated in order to analyze HCV evolutionary relationships between Uruguayan, Brazilian, and worldwide strains. For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2.", "For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2. 2.5. NS5A and NS5B Sequence Analysis.", "2.5. NS5A and NS5B Sequence Analysis. In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide.", "In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide. For this purpose, NS5A gene sequences corresponding to subtypes 1a (n = 160) and 1b (n = 88) were retrieved from Los Alamos HCV sequence database and from the NIAID ViPR [21, 22] . Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively.", "Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively. Using Seqman program, implemented in DNAStar 5.01 package (DNASTAR, Madison, USA), a world consensus nucleotide sequences were generated for each gene and subtype. Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated.", "Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated. The amino acid sequences obtained were compared in order to explore the presence of RASs as well as the presence of polymorphisms at a RAS position (RAPs) in Uruguayan HCV strains. RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] .", "RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] . To study the genetic variability of NS5A and NS5B regions of HCV strains circulating in Uruguayan patients, sequences of these regions (accession numbers MH070029-MH070090) were aligned with corresponding sequences from 59 HCV strains isolated elsewhere, representing all genotypes and main subtypes (for strains included in these analyses, see Supplementary Material Table S1 ). Therefore, maximum likelihood phylogenetic trees were constructed.", "Therefore, maximum likelihood phylogenetic trees were constructed. The results of these studies are shown in Figure 1 All strains in the phylogenies were assigned according to their genotype, and each cluster was supported by very high bootstrap values for both analyzed regions. Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b.", "Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b. The results of NS5A (Figure 1 (a)) and NS5B (Figure 1 \n\nGenotype 1b phylogenetic analyses were concordant for both genomic regions in all 31 sequences, suggesting no recombination events between these regions. To further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ).", "To further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ). As was previously described, two distinct 1a clades (clades 1 and 2) were observed. Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] .", "Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] . Whereas NS5A Uruguayan strains (in red) did not cluster in a particular clade, rather, they grouped dispersedly within all major world clades. With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence).", "With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence). AA substitutions at positions previously found to be potentially associated with resistance to NS5A inhibitors, as well as polymorphisms at a RAS position, were identified. These results are summarized in Table 1 .", "These results are summarized in Table 1 . RASs to NS5A inhibitors (L31M and L31V) were identified in 2 strains out of 25 (8%) fully sequenced samples. RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q.", "RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q. Although these substitutions were not reported as resistant, some changes at these positions were previously described as RASs in subtype 1a, namely H58D and K24R [27, 28] . Finally, substitution E62D was found in one subtype 1a strain.", "Finally, substitution E62D was found in one subtype 1a strain. This change is considered as a secondary substitution because, although it does not confer resistance by itself, when combined with a known RAS it does. In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] .", "In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] . In addition, several polymorphisms that have not been previously reported to be associated with a resistant phenotype were also detected (see Supplementary Material Table S3 ). In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences.", "In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences. Almost full-length AA sequences were obtained in 26 out of 31 analyzed strains. 23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein.", "23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein. This issue limited our studies, since many of the described RASs are observed as of residue 553. Importantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%).", "Importantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%). C451R was found in two isolates while A421V was found in only one. In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed.", "In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed. Finally, we found two RAPs: A421V (in 2 subtype 1b strains) and A553G (in 1 subtype 1a strain). Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] .", "Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] . In position 553, the substitution reported as resistant was A553T [8] . As was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ).", "As was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ). The advent of DAAs therapies constitutes one of the major breakthroughs in HCV infected patients management. However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems.", "However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems. The main limiting factors for worldwide access to DAAs in our region concern the high cost, the inadequate management of public healthcare systems, the limited access of low-income or uninsured populations to healthcare providers, and the lack of accurate epidemiological information [20, [30] [31] [32] . In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases.", "In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases. Despite the high rates of viral response achieved with DAA-based treatments, still 1 to10% of the patients fails to eliminate infection, and in these cases, baseline and emergent resistance variants turn out to be key factors contributing to treatment failure [5, 17, 33] . Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating.", "Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating. These facts could compromise the effectiveness of these new therapies in our country. We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] .", "We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] . The present study describes the prevalence of baseline NS5A and NS5B RASs in HCV genotype 1 infected DAA-naïve patients in a Uruguayan cohort. The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] .", "The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] . However, NS5A sequences from South America are poorly analyzed yet [9, 44] . Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] .", "Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] . Importantly, the prevalence and type of baseline NS5A RASs varies slightly by geographic regions. For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] .", "For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] . For this reason, we believe that there is a need to contribute data from our region, for which we still do not have enough information, apart from Brazil [9, 44] . The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ).", "The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ). L31M substitution confers resistance to daclatasvir (DCV), ledipasvir (LDV), and elbasvir (EBV) in both 1a and 1b subtypes [5, 6, 8, 28, 47, 48] , whereas substitution L31V does it to DCV in subtypes 1a and 1b, to LDV in subtype 1b, and to EBV in subtype 1a [5, 6, 28] . Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] .", "Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] . The substitutions H58P and K24Q found in two patients are considered as resistance-associated polymorphisms (RAPs). The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] .", "The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] . The substitution H58P was found as a baseline RAP in relapsers to LDV (HARVONI prescription, media/files/pdfs/medicines/liver-disease/harvoni/harvoni_pi. pdf?la=en).", "pdf?la=en). However, it is sometimes regarded as a RAS [10, 51] , despite conferring only 1.2 fold change in resistance in in vitro studies using the 1a replicon system [39] . We did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] .", "We did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] . The amino acid substitution E62H was found in one Uruguayan patient. Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] .", "Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] . The presence of baseline NS5A RASs impacts treatment outcome in some patient groups by affecting SVR rates. The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use.", "The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use. Regarding NS5B gene, global analysis (with the exception of South America [17, 19] ) revealed that NS5B DAA resistance substitutions are infrequent [14] . Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%).", "Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%). Substitutions found in this work were A421V and S556G associated in subtype 1a with resistance to BCV and dasabuvir (DSV), respectively [8, 28, 29, 54, 55] , and Q556R associated with resistance to DSV both in genotype 1a and 1b [12, 28] . Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV.", "Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV. In these cases, it appeared in combination with G558R (Trial Coral I-Cohort 2: RAPs in positions 421 and 553 (A421V in two subtype 1b isolates and A553G in one subtype 1b isolate) were also found. Although A421V has been associated with resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] .", "Although A421V has been associated with resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] . In position 553, the substitutions reported as resistant are A553T in subtype 1a [8] and A553V in subtype 1b [54] , conferring resistance to DSV. In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ).", "In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ). Nevertheless, substitution A421V was found in Brazil [17] , Argentina [19] , and Uruguay. The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] .", "The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] . Our findings further confirm and complement previous studies which evidenced a low prevalence of this substitution in vivo, probably due to its low replicative fitness [14, 18, 57] . Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] .", "Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] . These results show both diversity in the baseline polymorphisms found in different Latin American countries and in the evolutionary relationships of Uruguayan isolates ( Figure 2 ). This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host.", "This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host. It is worth mentioning that we live in a vast continent inhabited by populations with different genotypic characteristics that might, depending on the situation, require different approaches to treatment. Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] .", "Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] . Altogether, we believe that it could be important to carry out studies throughout the South American region in order to establish the prevalence of RASs in NS5A and NS5B in different countries. In fact, this will aid in understanding that not every treatment regimen might be adequate for every patient and country.", "In fact, this will aid in understanding that not every treatment regimen might be adequate for every patient and country. The data we presented here might guide not only physicians in making therapeutic decisions but also public health authorities in approving more diverse treatment combinations. These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population.", "These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population. To our knowledge, the present study revealed for the first time the presence of RASs in the NS5A and NS5B regions of HCV genotype 1 Uruguayan strains from patients who have not been previously treated with DAAs and is one of the few South American countries to report on this matter. It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure.", "It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure. However, individualized DAA therapy based on baseline resistance analysis may be beneficial for optimizing treatment efficacy in patients with HCV genotype 1 infection and risk factors for treatment failure. Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions.", "Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions. The data used to support the findings of this study are included within the article. The authors declare that they have no conflicts of interest. Fabián Aldunate and Natalia Echeverría contributed equally to this work.", "Fabián Aldunate and Natalia Echeverría contributed equally to this work. Supplementary Material Table S1 : hepatitis C Virus NS5A and NS5B sequences used as representatives of each genotype to perform the phylogenetic analysis. Their corresponding genotype, country of isolation, and GenBank accession number are indicated.", "Their corresponding genotype, country of isolation, and GenBank accession number are indicated. Supplementary Material Table S2 : hepatitis C Virus NS5A subtype 1a sequences used to reveal evolutionary relationships between Uruguayan strains and others isolated elsewhere. Their corresponding country of isolation and GenBank accession number are indicated.", "Their corresponding country of isolation and GenBank accession number are indicated. Supplementary Material Table S3 : amino acid substitutions in NS5A protein not previously associated with resistance to NS5A inhibitors. Supplementary Material Table S4 : amino acid substitutions in NS5B protein not previously associated with resistance to polymerase inhibitors. (Supplementary Materials)" ]

[ "Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction.", "Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries.", "The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil.", "Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity. Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs).", "Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs). These antivirals have significantly increased response rates (up to 98%) and greatly reduced treatment duration [1] . Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] .", "Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] . However, the extreme mutation and high replication rates of HCV, together with the immune system pressure, lead to a remarkable genetic variability that can compromise the high response rates to DAAs due to the preexistence of resistanceassociated substitutions (RASs) [5, 6] . Each drug or class of DAA is characterized by specific resistance profiles.", "Each drug or class of DAA is characterized by specific resistance profiles. The likelihood that a DAA will select for and allow outgrowth of viral populations carrying RASs depends on the DAA's genetic barrier to resistance (the number and type of mutations needed to generate an amino acid substitution that confers resistance), the viral fitness (replicative capacity) of the resistant variant, and viral genotypes and subtypes [7, 8] . The prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] .", "The prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] . However, apart from Brazil and Argentina, this issue has not been fully addressed in other South American countries yet [9, [17] [18] [19] . The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] .", "The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] . In this study, we explored the presence of resistance variants to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients chronically infected with hepatitis C. Here, we aimed to contribute to the knowledge of the circulation of HCV resistant variants in the South American region. Samples.", "Samples. Serum samples were obtained from 31 patients with serological markers for HCV, which were recruited between 2015 and 2017 at the Gastroenterology Clinic from Hospital de Clínicas, Montevideo, Uruguay. HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA).", "HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA). Patients selected for this study were both chronically infected with HCV genotype 1 and DAA treatment-naïve at the time of blood extraction. Written informed consent was obtained from all patients.", "Written informed consent was obtained from all patients. The studies have been performed according to the World Medical Association Declaration of Helsinki and approved by the appropriate institutional board (Hospital de Clínicas ethical committee). 2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification.", "2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification. Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol.", "Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol. The viral RNA was heated at 65°C for 5 min and used as a template for a reverse transcription reaction. The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA).", "The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA). The reverse transcription was performed at 42°C for 50 min, and then the reverse transcriptase enzyme was inactivated at 70°C for 15 min. PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously described [10] .", "PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously described [10] . Amplicons were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Science, Buckinghamshire, UK) according to the manufacturer's protocol. 2.3. NS5A and NS5B Sequencing.", "2.3. NS5A and NS5B Sequencing. The purified product was then sequenced using the same sets of primers used for PCR amplification. Bidirectional Sanger sequencing was performed by Macrogen Korea (\n\n2.4. NS5A and NS5B Genotype Determination.", "NS5A and NS5B Genotype Determination. HCV NS5A and NS5B consensus sequences obtained from Uruguayan patients were aligned with sequences from HCV representing all genotypes and main subtypes isolated in different geographic regions of the world. These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] .", "These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] . For strains included in these studies, see Supplementary Material Table S1 . Sequences were aligned using the CLUSTAL W software [23] .", "Sequences were aligned using the CLUSTAL W software [23] . Once aligned, the best evolutionary model that described our sequence data was assessed using ModelGenerator program [24] . Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] .", "Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] . For NS5A, 953 nucleotides (positions 6367 to 7319, relative to HCV 1a reference strain, H77 NC_004102) were included in the phylogenetic analysis, whereas for NS5B, only 361 nucleotides corresponding to the Okamoto region (positions 8265 to 8625, relative to strain H77 NC_004102) were included. As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates).", "As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates). For NS5A 1a Uruguayan sequences (n = 20), a second alignment and maximum likelihood phylogenetic tree was generated in order to analyze HCV evolutionary relationships between Uruguayan, Brazilian, and worldwide strains. For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2.", "For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2. 2.5. NS5A and NS5B Sequence Analysis.", "2.5. NS5A and NS5B Sequence Analysis. In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide.", "In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide. For this purpose, NS5A gene sequences corresponding to subtypes 1a (n = 160) and 1b (n = 88) were retrieved from Los Alamos HCV sequence database and from the NIAID ViPR [21, 22] . Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively.", "Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively. Using Seqman program, implemented in DNAStar 5.01 package (DNASTAR, Madison, USA), a world consensus nucleotide sequences were generated for each gene and subtype. Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated.", "Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated. The amino acid sequences obtained were compared in order to explore the presence of RASs as well as the presence of polymorphisms at a RAS position (RAPs) in Uruguayan HCV strains. RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] .", "RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] . To study the genetic variability of NS5A and NS5B regions of HCV strains circulating in Uruguayan patients, sequences of these regions (accession numbers MH070029-MH070090) were aligned with corresponding sequences from 59 HCV strains isolated elsewhere, representing all genotypes and main subtypes (for strains included in these analyses, see Supplementary Material Table S1 ). Therefore, maximum likelihood phylogenetic trees were constructed.", "Therefore, maximum likelihood phylogenetic trees were constructed. The results of these studies are shown in Figure 1 All strains in the phylogenies were assigned according to their genotype, and each cluster was supported by very high bootstrap values for both analyzed regions. Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b.", "Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b. The results of NS5A (Figure 1 (a)) and NS5B (Figure 1 \n\nGenotype 1b phylogenetic analyses were concordant for both genomic regions in all 31 sequences, suggesting no recombination events between these regions. To further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ).", "To further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ). As was previously described, two distinct 1a clades (clades 1 and 2) were observed. Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] .", "Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] . Whereas NS5A Uruguayan strains (in red) did not cluster in a particular clade, rather, they grouped dispersedly within all major world clades. With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence).", "With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence). AA substitutions at positions previously found to be potentially associated with resistance to NS5A inhibitors, as well as polymorphisms at a RAS position, were identified. These results are summarized in Table 1 .", "These results are summarized in Table 1 . RASs to NS5A inhibitors (L31M and L31V) were identified in 2 strains out of 25 (8%) fully sequenced samples. RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q.", "RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q. Although these substitutions were not reported as resistant, some changes at these positions were previously described as RASs in subtype 1a, namely H58D and K24R [27, 28] . Finally, substitution E62D was found in one subtype 1a strain.", "Finally, substitution E62D was found in one subtype 1a strain. This change is considered as a secondary substitution because, although it does not confer resistance by itself, when combined with a known RAS it does. In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] .", "In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] . In addition, several polymorphisms that have not been previously reported to be associated with a resistant phenotype were also detected (see Supplementary Material Table S3 ). In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences.", "In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences. Almost full-length AA sequences were obtained in 26 out of 31 analyzed strains. 23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein.", "23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein. This issue limited our studies, since many of the described RASs are observed as of residue 553. Importantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%).", "Importantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%). C451R was found in two isolates while A421V was found in only one. In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed.", "In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed. Finally, we found two RAPs: A421V (in 2 subtype 1b strains) and A553G (in 1 subtype 1a strain). Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] .", "Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] . In position 553, the substitution reported as resistant was A553T [8] . As was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ).", "As was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ). The advent of DAAs therapies constitutes one of the major breakthroughs in HCV infected patients management. However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems.", "However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems. The main limiting factors for worldwide access to DAAs in our region concern the high cost, the inadequate management of public healthcare systems, the limited access of low-income or uninsured populations to healthcare providers, and the lack of accurate epidemiological information [20, [30] [31] [32] . In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases.", "In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases. Despite the high rates of viral response achieved with DAA-based treatments, still 1 to10% of the patients fails to eliminate infection, and in these cases, baseline and emergent resistance variants turn out to be key factors contributing to treatment failure [5, 17, 33] . Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating.", "Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating. These facts could compromise the effectiveness of these new therapies in our country. We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] .", "We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] . The present study describes the prevalence of baseline NS5A and NS5B RASs in HCV genotype 1 infected DAA-naïve patients in a Uruguayan cohort. The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] .", "The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] . However, NS5A sequences from South America are poorly analyzed yet [9, 44] . Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] .", "Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] . Importantly, the prevalence and type of baseline NS5A RASs varies slightly by geographic regions. For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] .", "For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] . For this reason, we believe that there is a need to contribute data from our region, for which we still do not have enough information, apart from Brazil [9, 44] . The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ).", "The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ). L31M substitution confers resistance to daclatasvir (DCV), ledipasvir (LDV), and elbasvir (EBV) in both 1a and 1b subtypes [5, 6, 8, 28, 47, 48] , whereas substitution L31V does it to DCV in subtypes 1a and 1b, to LDV in subtype 1b, and to EBV in subtype 1a [5, 6, 28] . Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] .", "Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] . The substitutions H58P and K24Q found in two patients are considered as resistance-associated polymorphisms (RAPs). The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] .", "The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] . The substitution H58P was found as a baseline RAP in relapsers to LDV (HARVONI prescription, media/files/pdfs/medicines/liver-disease/harvoni/harvoni_pi. pdf?la=en).", "pdf?la=en). However, it is sometimes regarded as a RAS [10, 51] , despite conferring only 1.2 fold change in resistance in in vitro studies using the 1a replicon system [39] . We did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] .", "We did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] . The amino acid substitution E62H was found in one Uruguayan patient. Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] .", "Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] . The presence of baseline NS5A RASs impacts treatment outcome in some patient groups by affecting SVR rates. The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use.", "The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use. Regarding NS5B gene, global analysis (with the exception of South America [17, 19] ) revealed that NS5B DAA resistance substitutions are infrequent [14] . Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%).", "Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%). Substitutions found in this work were A421V and S556G associated in subtype 1a with resistance to BCV and dasabuvir (DSV), respectively [8, 28, 29, 54, 55] , and Q556R associated with resistance to DSV both in genotype 1a and 1b [12, 28] . Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV.", "Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV. In these cases, it appeared in combination with G558R (Trial Coral I-Cohort 2: RAPs in positions 421 and 553 (A421V in two subtype 1b isolates and A553G in one subtype 1b isolate) were also found. Although A421V has been associated with resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] .", "Although A421V has been associated with resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] . In position 553, the substitutions reported as resistant are A553T in subtype 1a [8] and A553V in subtype 1b [54] , conferring resistance to DSV. In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ).", "In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ). Nevertheless, substitution A421V was found in Brazil [17] , Argentina [19] , and Uruguay. The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] .", "The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] . Our findings further confirm and complement previous studies which evidenced a low prevalence of this substitution in vivo, probably due to its low replicative fitness [14, 18, 57] . Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] .", "Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] . These results show both diversity in the baseline polymorphisms found in different Latin American countries and in the evolutionary relationships of Uruguayan isolates ( Figure 2 ). This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host.", "This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host. It is worth mentioning that we live in a vast continent inhabited by populations with different genotypic characteristics that might, depending on the situation, require different approaches to treatment. Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] .", "Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] . Altogether, we believe that it could be important to carry out studies throughout the South American region in order to establish the prevalence of RASs in NS5A and NS5B in different countries. In fact, this will aid in understanding that not every treatment regimen might be adequate for every patient and country.", "In fact, this will aid in understanding that not every treatment regimen might be adequate for every patient and country. The data we presented here might guide not only physicians in making therapeutic decisions but also public health authorities in approving more diverse treatment combinations. These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population.", "These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population. To our knowledge, the present study revealed for the first time the presence of RASs in the NS5A and NS5B regions of HCV genotype 1 Uruguayan strains from patients who have not been previously treated with DAAs and is one of the few South American countries to report on this matter. It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure.", "It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure. However, individualized DAA therapy based on baseline resistance analysis may be beneficial for optimizing treatment efficacy in patients with HCV genotype 1 infection and risk factors for treatment failure. Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions.", "Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions. The data used to support the findings of this study are included within the article. The authors declare that they have no conflicts of interest. Fabián Aldunate and Natalia Echeverría contributed equally to this work.", "Fabián Aldunate and Natalia Echeverría contributed equally to this work. Supplementary Material Table S1 : hepatitis C Virus NS5A and NS5B sequences used as representatives of each genotype to perform the phylogenetic analysis. Their corresponding genotype, country of isolation, and GenBank accession number are indicated.", "Their corresponding genotype, country of isolation, and GenBank accession number are indicated. Supplementary Material Table S2 : hepatitis C Virus NS5A subtype 1a sequences used to reveal evolutionary relationships between Uruguayan strains and others isolated elsewhere. Their corresponding country of isolation and GenBank accession number are indicated.", "Their corresponding country of isolation and GenBank accession number are indicated. Supplementary Material Table S3 : amino acid substitutions in NS5A protein not previously associated with resistance to NS5A inhibitors. Supplementary Material Table S4 : amino acid substitutions in NS5B protein not previously associated with resistance to polymerase inhibitors. (Supplementary Materials)" ]

[ "Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction.", "Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries.", "The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil.", "Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity. Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs).", "Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs). These antivirals have significantly increased response rates (up to 98%) and greatly reduced treatment duration [1] . Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] .", "Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] . However, the extreme mutation and high replication rates of HCV, together with the immune system pressure, lead to a remarkable genetic variability that can compromise the high response rates to DAAs due to the preexistence of resistanceassociated substitutions (RASs) [5, 6] . Each drug or class of DAA is characterized by specific resistance profiles.", "Each drug or class of DAA is characterized by specific resistance profiles. The likelihood that a DAA will select for and allow outgrowth of viral populations carrying RASs depends on the DAA's genetic barrier to resistance (the number and type of mutations needed to generate an amino acid substitution that confers resistance), the viral fitness (replicative capacity) of the resistant variant, and viral genotypes and subtypes [7, 8] . The prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] .", "The prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] . However, apart from Brazil and Argentina, this issue has not been fully addressed in other South American countries yet [9, [17] [18] [19] . The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] .", "The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] . In this study, we explored the presence of resistance variants to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients chronically infected with hepatitis C. Here, we aimed to contribute to the knowledge of the circulation of HCV resistant variants in the South American region. Samples.", "Samples. Serum samples were obtained from 31 patients with serological markers for HCV, which were recruited between 2015 and 2017 at the Gastroenterology Clinic from Hospital de Clínicas, Montevideo, Uruguay. HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA).", "HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA). Patients selected for this study were both chronically infected with HCV genotype 1 and DAA treatment-naïve at the time of blood extraction. Written informed consent was obtained from all patients.", "Written informed consent was obtained from all patients. The studies have been performed according to the World Medical Association Declaration of Helsinki and approved by the appropriate institutional board (Hospital de Clínicas ethical committee). 2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification.", "2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification. Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol.", "Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol. The viral RNA was heated at 65°C for 5 min and used as a template for a reverse transcription reaction. The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA).", "The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA). The reverse transcription was performed at 42°C for 50 min, and then the reverse transcriptase enzyme was inactivated at 70°C for 15 min. PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously described [10] .", "PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously described [10] . Amplicons were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Science, Buckinghamshire, UK) according to the manufacturer's protocol. 2.3. NS5A and NS5B Sequencing.", "2.3. NS5A and NS5B Sequencing. The purified product was then sequenced using the same sets of primers used for PCR amplification. Bidirectional Sanger sequencing was performed by Macrogen Korea (\n\n2.4. NS5A and NS5B Genotype Determination.", "NS5A and NS5B Genotype Determination. HCV NS5A and NS5B consensus sequences obtained from Uruguayan patients were aligned with sequences from HCV representing all genotypes and main subtypes isolated in different geographic regions of the world. These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] .", "These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] . For strains included in these studies, see Supplementary Material Table S1 . Sequences were aligned using the CLUSTAL W software [23] .", "Sequences were aligned using the CLUSTAL W software [23] . Once aligned, the best evolutionary model that described our sequence data was assessed using ModelGenerator program [24] . Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] .", "Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] . For NS5A, 953 nucleotides (positions 6367 to 7319, relative to HCV 1a reference strain, H77 NC_004102) were included in the phylogenetic analysis, whereas for NS5B, only 361 nucleotides corresponding to the Okamoto region (positions 8265 to 8625, relative to strain H77 NC_004102) were included. As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates).", "As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates). For NS5A 1a Uruguayan sequences (n = 20), a second alignment and maximum likelihood phylogenetic tree was generated in order to analyze HCV evolutionary relationships between Uruguayan, Brazilian, and worldwide strains. For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2.", "For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2. 2.5. NS5A and NS5B Sequence Analysis.", "2.5. NS5A and NS5B Sequence Analysis. In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide.", "In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide. For this purpose, NS5A gene sequences corresponding to subtypes 1a (n = 160) and 1b (n = 88) were retrieved from Los Alamos HCV sequence database and from the NIAID ViPR [21, 22] . Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively.", "Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively. Using Seqman program, implemented in DNAStar 5.01 package (DNASTAR, Madison, USA), a world consensus nucleotide sequences were generated for each gene and subtype. Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated.", "Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated. The amino acid sequences obtained were compared in order to explore the presence of RASs as well as the presence of polymorphisms at a RAS position (RAPs) in Uruguayan HCV strains. RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] .", "RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] . To study the genetic variability of NS5A and NS5B regions of HCV strains circulating in Uruguayan patients, sequences of these regions (accession numbers MH070029-MH070090) were aligned with corresponding sequences from 59 HCV strains isolated elsewhere, representing all genotypes and main subtypes (for strains included in these analyses, see Supplementary Material Table S1 ). Therefore, maximum likelihood phylogenetic trees were constructed.", "Therefore, maximum likelihood phylogenetic trees were constructed. The results of these studies are shown in Figure 1 All strains in the phylogenies were assigned according to their genotype, and each cluster was supported by very high bootstrap values for both analyzed regions. Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b.", "Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b. The results of NS5A (Figure 1 (a)) and NS5B (Figure 1 \n\nGenotype 1b phylogenetic analyses were concordant for both genomic regions in all 31 sequences, suggesting no recombination events between these regions. To further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ).", "To further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ). As was previously described, two distinct 1a clades (clades 1 and 2) were observed. Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] .", "Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] . Whereas NS5A Uruguayan strains (in red) did not cluster in a particular clade, rather, they grouped dispersedly within all major world clades. With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence).", "With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence). AA substitutions at positions previously found to be potentially associated with resistance to NS5A inhibitors, as well as polymorphisms at a RAS position, were identified. These results are summarized in Table 1 .", "These results are summarized in Table 1 . RASs to NS5A inhibitors (L31M and L31V) were identified in 2 strains out of 25 (8%) fully sequenced samples. RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q.", "RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q. Although these substitutions were not reported as resistant, some changes at these positions were previously described as RASs in subtype 1a, namely H58D and K24R [27, 28] . Finally, substitution E62D was found in one subtype 1a strain.", "Finally, substitution E62D was found in one subtype 1a strain. This change is considered as a secondary substitution because, although it does not confer resistance by itself, when combined with a known RAS it does. In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] .", "In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] . In addition, several polymorphisms that have not been previously reported to be associated with a resistant phenotype were also detected (see Supplementary Material Table S3 ). In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences.", "In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences. Almost full-length AA sequences were obtained in 26 out of 31 analyzed strains. 23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein.", "23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein. This issue limited our studies, since many of the described RASs are observed as of residue 553. Importantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%).", "Importantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%). C451R was found in two isolates while A421V was found in only one. In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed.", "In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed. Finally, we found two RAPs: A421V (in 2 subtype 1b strains) and A553G (in 1 subtype 1a strain). Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] .", "Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] . In position 553, the substitution reported as resistant was A553T [8] . As was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ).", "As was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ). The advent of DAAs therapies constitutes one of the major breakthroughs in HCV infected patients management. However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems.", "However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems. The main limiting factors for worldwide access to DAAs in our region concern the high cost, the inadequate management of public healthcare systems, the limited access of low-income or uninsured populations to healthcare providers, and the lack of accurate epidemiological information [20, [30] [31] [32] . In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases.", "In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases. Despite the high rates of viral response achieved with DAA-based treatments, still 1 to10% of the patients fails to eliminate infection, and in these cases, baseline and emergent resistance variants turn out to be key factors contributing to treatment failure [5, 17, 33] . Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating.", "Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating. These facts could compromise the effectiveness of these new therapies in our country. We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] .", "We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] . The present study describes the prevalence of baseline NS5A and NS5B RASs in HCV genotype 1 infected DAA-naïve patients in a Uruguayan cohort. The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] .", "The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] . However, NS5A sequences from South America are poorly analyzed yet [9, 44] . Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] .", "Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] . Importantly, the prevalence and type of baseline NS5A RASs varies slightly by geographic regions. For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] .", "For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] . For this reason, we believe that there is a need to contribute data from our region, for which we still do not have enough information, apart from Brazil [9, 44] . The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ).", "The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ). L31M substitution confers resistance to daclatasvir (DCV), ledipasvir (LDV), and elbasvir (EBV) in both 1a and 1b subtypes [5, 6, 8, 28, 47, 48] , whereas substitution L31V does it to DCV in subtypes 1a and 1b, to LDV in subtype 1b, and to EBV in subtype 1a [5, 6, 28] . Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] .", "Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] . The substitutions H58P and K24Q found in two patients are considered as resistance-associated polymorphisms (RAPs). The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] .", "The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] . The substitution H58P was found as a baseline RAP in relapsers to LDV (HARVONI prescription, media/files/pdfs/medicines/liver-disease/harvoni/harvoni_pi. pdf?la=en).", "pdf?la=en). However, it is sometimes regarded as a RAS [10, 51] , despite conferring only 1.2 fold change in resistance in in vitro studies using the 1a replicon system [39] . We did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] .", "We did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] . The amino acid substitution E62H was found in one Uruguayan patient. Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] .", "Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] . The presence of baseline NS5A RASs impacts treatment outcome in some patient groups by affecting SVR rates. The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use.", "The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use. Regarding NS5B gene, global analysis (with the exception of South America [17, 19] ) revealed that NS5B DAA resistance substitutions are infrequent [14] . Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%).", "Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%). Substitutions found in this work were A421V and S556G associated in subtype 1a with resistance to BCV and dasabuvir (DSV), respectively [8, 28, 29, 54, 55] , and Q556R associated with resistance to DSV both in genotype 1a and 1b [12, 28] . Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV.", "Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV. In these cases, it appeared in combination with G558R (Trial Coral I-Cohort 2: RAPs in positions 421 and 553 (A421V in two subtype 1b isolates and A553G in one subtype 1b isolate) were also found. Although A421V has been associated with resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] .", "Although A421V has been associated with resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] . In position 553, the substitutions reported as resistant are A553T in subtype 1a [8] and A553V in subtype 1b [54] , conferring resistance to DSV. In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ).", "In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ). Nevertheless, substitution A421V was found in Brazil [17] , Argentina [19] , and Uruguay. The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] .", "The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] . Our findings further confirm and complement previous studies which evidenced a low prevalence of this substitution in vivo, probably due to its low replicative fitness [14, 18, 57] . Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] .", "Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] . These results show both diversity in the baseline polymorphisms found in different Latin American countries and in the evolutionary relationships of Uruguayan isolates ( Figure 2 ). This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host.", "This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host. It is worth mentioning that we live in a vast continent inhabited by populations with different genotypic characteristics that might, depending on the situation, require different approaches to treatment. Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] .", "Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] . Altogether, we believe that it could be important to carry out studies throughout the South American region in order to establish the prevalence of RASs in NS5A and NS5B in different countries. In fact, this will aid in understanding that not every treatment regimen might be adequate for every patient and country.", "In fact, this will aid in understanding that not every treatment regimen might be adequate for every patient and country. The data we presented here might guide not only physicians in making therapeutic decisions but also public health authorities in approving more diverse treatment combinations. These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population.", "These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population. To our knowledge, the present study revealed for the first time the presence of RASs in the NS5A and NS5B regions of HCV genotype 1 Uruguayan strains from patients who have not been previously treated with DAAs and is one of the few South American countries to report on this matter. It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure.", "It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure. However, individualized DAA therapy based on baseline resistance analysis may be beneficial for optimizing treatment efficacy in patients with HCV genotype 1 infection and risk factors for treatment failure. Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions.", "Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions. The data used to support the findings of this study are included within the article. The authors declare that they have no conflicts of interest. Fabián Aldunate and Natalia Echeverría contributed equally to this work.", "Fabián Aldunate and Natalia Echeverría contributed equally to this work. Supplementary Material Table S1 : hepatitis C Virus NS5A and NS5B sequences used as representatives of each genotype to perform the phylogenetic analysis. Their corresponding genotype, country of isolation, and GenBank accession number are indicated.", "Their corresponding genotype, country of isolation, and GenBank accession number are indicated. Supplementary Material Table S2 : hepatitis C Virus NS5A subtype 1a sequences used to reveal evolutionary relationships between Uruguayan strains and others isolated elsewhere. Their corresponding country of isolation and GenBank accession number are indicated.", "Their corresponding country of isolation and GenBank accession number are indicated. Supplementary Material Table S3 : amino acid substitutions in NS5A protein not previously associated with resistance to NS5A inhibitors. Supplementary Material Table S4 : amino acid substitutions in NS5B protein not previously associated with resistance to polymerase inhibitors. (Supplementary Materials)" ]

[ "Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction.", "Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries.", "The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil.", "Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity. Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs).", "Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs). These antivirals have significantly increased response rates (up to 98%) and greatly reduced treatment duration [1] . Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] .", "Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] . However, the extreme mutation and high replication rates of HCV, together with the immune system pressure, lead to a remarkable genetic variability that can compromise the high response rates to DAAs due to the preexistence of resistanceassociated substitutions (RASs) [5, 6] . Each drug or class of DAA is characterized by specific resistance profiles.", "Each drug or class of DAA is characterized by specific resistance profiles. The likelihood that a DAA will select for and allow outgrowth of viral populations carrying RASs depends on the DAA's genetic barrier to resistance (the number and type of mutations needed to generate an amino acid substitution that confers resistance), the viral fitness (replicative capacity) of the resistant variant, and viral genotypes and subtypes [7, 8] . The prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] .", "The prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] . However, apart from Brazil and Argentina, this issue has not been fully addressed in other South American countries yet [9, [17] [18] [19] . The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] .", "The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] . In this study, we explored the presence of resistance variants to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients chronically infected with hepatitis C. Here, we aimed to contribute to the knowledge of the circulation of HCV resistant variants in the South American region. Samples.", "Samples. Serum samples were obtained from 31 patients with serological markers for HCV, which were recruited between 2015 and 2017 at the Gastroenterology Clinic from Hospital de Clínicas, Montevideo, Uruguay. HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA).", "HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA). Patients selected for this study were both chronically infected with HCV genotype 1 and DAA treatment-naïve at the time of blood extraction. Written informed consent was obtained from all patients.", "Written informed consent was obtained from all patients. The studies have been performed according to the World Medical Association Declaration of Helsinki and approved by the appropriate institutional board (Hospital de Clínicas ethical committee). 2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification.", "2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification. Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol.", "Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol. The viral RNA was heated at 65°C for 5 min and used as a template for a reverse transcription reaction. The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA).", "The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA). The reverse transcription was performed at 42°C for 50 min, and then the reverse transcriptase enzyme was inactivated at 70°C for 15 min. PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously described [10] .", "PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously described [10] . Amplicons were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Science, Buckinghamshire, UK) according to the manufacturer's protocol. 2.3. NS5A and NS5B Sequencing.", "2.3. NS5A and NS5B Sequencing. The purified product was then sequenced using the same sets of primers used for PCR amplification. Bidirectional Sanger sequencing was performed by Macrogen Korea (\n\n2.4. NS5A and NS5B Genotype Determination.", "NS5A and NS5B Genotype Determination. HCV NS5A and NS5B consensus sequences obtained from Uruguayan patients were aligned with sequences from HCV representing all genotypes and main subtypes isolated in different geographic regions of the world. These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] .", "These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] . For strains included in these studies, see Supplementary Material Table S1 . Sequences were aligned using the CLUSTAL W software [23] .", "Sequences were aligned using the CLUSTAL W software [23] . Once aligned, the best evolutionary model that described our sequence data was assessed using ModelGenerator program [24] . Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] .", "Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] . For NS5A, 953 nucleotides (positions 6367 to 7319, relative to HCV 1a reference strain, H77 NC_004102) were included in the phylogenetic analysis, whereas for NS5B, only 361 nucleotides corresponding to the Okamoto region (positions 8265 to 8625, relative to strain H77 NC_004102) were included. As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates).", "As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates). For NS5A 1a Uruguayan sequences (n = 20), a second alignment and maximum likelihood phylogenetic tree was generated in order to analyze HCV evolutionary relationships between Uruguayan, Brazilian, and worldwide strains. For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2.", "For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2. 2.5. NS5A and NS5B Sequence Analysis.", "2.5. NS5A and NS5B Sequence Analysis. In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide.", "In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide. For this purpose, NS5A gene sequences corresponding to subtypes 1a (n = 160) and 1b (n = 88) were retrieved from Los Alamos HCV sequence database and from the NIAID ViPR [21, 22] . Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively.", "Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively. Using Seqman program, implemented in DNAStar 5.01 package (DNASTAR, Madison, USA), a world consensus nucleotide sequences were generated for each gene and subtype. Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated.", "Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated. The amino acid sequences obtained were compared in order to explore the presence of RASs as well as the presence of polymorphisms at a RAS position (RAPs) in Uruguayan HCV strains. RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] .", "RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] . To study the genetic variability of NS5A and NS5B regions of HCV strains circulating in Uruguayan patients, sequences of these regions (accession numbers MH070029-MH070090) were aligned with corresponding sequences from 59 HCV strains isolated elsewhere, representing all genotypes and main subtypes (for strains included in these analyses, see Supplementary Material Table S1 ). Therefore, maximum likelihood phylogenetic trees were constructed.", "Therefore, maximum likelihood phylogenetic trees were constructed. The results of these studies are shown in Figure 1 All strains in the phylogenies were assigned according to their genotype, and each cluster was supported by very high bootstrap values for both analyzed regions. Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b.", "Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b. The results of NS5A (Figure 1 (a)) and NS5B (Figure 1 \n\nGenotype 1b phylogenetic analyses were concordant for both genomic regions in all 31 sequences, suggesting no recombination events between these regions. To further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ).", "To further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ). As was previously described, two distinct 1a clades (clades 1 and 2) were observed. Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] .", "Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] . Whereas NS5A Uruguayan strains (in red) did not cluster in a particular clade, rather, they grouped dispersedly within all major world clades. With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence).", "With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence). AA substitutions at positions previously found to be potentially associated with resistance to NS5A inhibitors, as well as polymorphisms at a RAS position, were identified. These results are summarized in Table 1 .", "These results are summarized in Table 1 . RASs to NS5A inhibitors (L31M and L31V) were identified in 2 strains out of 25 (8%) fully sequenced samples. RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q.", "RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q. Although these substitutions were not reported as resistant, some changes at these positions were previously described as RASs in subtype 1a, namely H58D and K24R [27, 28] . Finally, substitution E62D was found in one subtype 1a strain.", "Finally, substitution E62D was found in one subtype 1a strain. This change is considered as a secondary substitution because, although it does not confer resistance by itself, when combined with a known RAS it does. In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] .", "In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] . In addition, several polymorphisms that have not been previously reported to be associated with a resistant phenotype were also detected (see Supplementary Material Table S3 ). In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences.", "In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences. Almost full-length AA sequences were obtained in 26 out of 31 analyzed strains. 23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein.", "23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein. This issue limited our studies, since many of the described RASs are observed as of residue 553. Importantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%).", "Importantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%). C451R was found in two isolates while A421V was found in only one. In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed.", "In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed. Finally, we found two RAPs: A421V (in 2 subtype 1b strains) and A553G (in 1 subtype 1a strain). Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] .", "Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] . In position 553, the substitution reported as resistant was A553T [8] . As was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ).", "As was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ). The advent of DAAs therapies constitutes one of the major breakthroughs in HCV infected patients management. However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems.", "However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems. The main limiting factors for worldwide access to DAAs in our region concern the high cost, the inadequate management of public healthcare systems, the limited access of low-income or uninsured populations to healthcare providers, and the lack of accurate epidemiological information [20, [30] [31] [32] . In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases.", "In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases. Despite the high rates of viral response achieved with DAA-based treatments, still 1 to10% of the patients fails to eliminate infection, and in these cases, baseline and emergent resistance variants turn out to be key factors contributing to treatment failure [5, 17, 33] . Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating.", "Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating. These facts could compromise the effectiveness of these new therapies in our country. We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] .", "We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] . The present study describes the prevalence of baseline NS5A and NS5B RASs in HCV genotype 1 infected DAA-naïve patients in a Uruguayan cohort. The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] .", "The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] . However, NS5A sequences from South America are poorly analyzed yet [9, 44] . Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] .", "Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] . Importantly, the prevalence and type of baseline NS5A RASs varies slightly by geographic regions. For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] .", "For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] . For this reason, we believe that there is a need to contribute data from our region, for which we still do not have enough information, apart from Brazil [9, 44] . The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ).", "The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ). L31M substitution confers resistance to daclatasvir (DCV), ledipasvir (LDV), and elbasvir (EBV) in both 1a and 1b subtypes [5, 6, 8, 28, 47, 48] , whereas substitution L31V does it to DCV in subtypes 1a and 1b, to LDV in subtype 1b, and to EBV in subtype 1a [5, 6, 28] . Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] .", "Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] . The substitutions H58P and K24Q found in two patients are considered as resistance-associated polymorphisms (RAPs). The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] .", "The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] . The substitution H58P was found as a baseline RAP in relapsers to LDV (HARVONI prescription, media/files/pdfs/medicines/liver-disease/harvoni/harvoni_pi. pdf?la=en).", "pdf?la=en). However, it is sometimes regarded as a RAS [10, 51] , despite conferring only 1.2 fold change in resistance in in vitro studies using the 1a replicon system [39] . We did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] .", "We did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] . The amino acid substitution E62H was found in one Uruguayan patient. Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] .", "Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] . The presence of baseline NS5A RASs impacts treatment outcome in some patient groups by affecting SVR rates. The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use.", "The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use. Regarding NS5B gene, global analysis (with the exception of South America [17, 19] ) revealed that NS5B DAA resistance substitutions are infrequent [14] . Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%).", "Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%). Substitutions found in this work were A421V and S556G associated in subtype 1a with resistance to BCV and dasabuvir (DSV), respectively [8, 28, 29, 54, 55] , and Q556R associated with resistance to DSV both in genotype 1a and 1b [12, 28] . Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV.", "Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV. In these cases, it appeared in combination with G558R (Trial Coral I-Cohort 2: RAPs in positions 421 and 553 (A421V in two subtype 1b isolates and A553G in one subtype 1b isolate) were also found. Although A421V has been associated with resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] .", "Although A421V has been associated with resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] . In position 553, the substitutions reported as resistant are A553T in subtype 1a [8] and A553V in subtype 1b [54] , conferring resistance to DSV. In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ).", "In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ). Nevertheless, substitution A421V was found in Brazil [17] , Argentina [19] , and Uruguay. The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] .", "The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] . Our findings further confirm and complement previous studies which evidenced a low prevalence of this substitution in vivo, probably due to its low replicative fitness [14, 18, 57] . Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] .", "Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] . These results show both diversity in the baseline polymorphisms found in different Latin American countries and in the evolutionary relationships of Uruguayan isolates ( Figure 2 ). This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host.", "This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host. It is worth mentioning that we live in a vast continent inhabited by populations with different genotypic characteristics that might, depending on the situation, require different approaches to treatment. Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] .", "Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] . Altogether, we believe that it could be important to carry out studies throughout the South American region in order to establish the prevalence of RASs in NS5A and NS5B in different countries. In fact, this will aid in understanding that not every treatment regimen might be adequate for every patient and country.", "In fact, this will aid in understanding that not every treatment regimen might be adequate for every patient and country. The data we presented here might guide not only physicians in making therapeutic decisions but also public health authorities in approving more diverse treatment combinations. These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population.", "These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population. To our knowledge, the present study revealed for the first time the presence of RASs in the NS5A and NS5B regions of HCV genotype 1 Uruguayan strains from patients who have not been previously treated with DAAs and is one of the few South American countries to report on this matter. It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure.", "It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure. However, individualized DAA therapy based on baseline resistance analysis may be beneficial for optimizing treatment efficacy in patients with HCV genotype 1 infection and risk factors for treatment failure. Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions.", "Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions. The data used to support the findings of this study are included within the article. The authors declare that they have no conflicts of interest. Fabián Aldunate and Natalia Echeverría contributed equally to this work.", "Fabián Aldunate and Natalia Echeverría contributed equally to this work. Supplementary Material Table S1 : hepatitis C Virus NS5A and NS5B sequences used as representatives of each genotype to perform the phylogenetic analysis. Their corresponding genotype, country of isolation, and GenBank accession number are indicated.", "Their corresponding genotype, country of isolation, and GenBank accession number are indicated. Supplementary Material Table S2 : hepatitis C Virus NS5A subtype 1a sequences used to reveal evolutionary relationships between Uruguayan strains and others isolated elsewhere. Their corresponding country of isolation and GenBank accession number are indicated.", "Their corresponding country of isolation and GenBank accession number are indicated. Supplementary Material Table S3 : amino acid substitutions in NS5A protein not previously associated with resistance to NS5A inhibitors. Supplementary Material Table S4 : amino acid substitutions in NS5B protein not previously associated with resistance to polymerase inhibitors. (Supplementary Materials)" ]

[ "Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction.", "Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries.", "The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil.", "Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity. Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs).", "Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs). These antivirals have significantly increased response rates (up to 98%) and greatly reduced treatment duration [1] . Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] .", "Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] . However, the extreme mutation and high replication rates of HCV, together with the immune system pressure, lead to a remarkable genetic variability that can compromise the high response rates to DAAs due to the preexistence of resistanceassociated substitutions (RASs) [5, 6] . Each drug or class of DAA is characterized by specific resistance profiles.", "Each drug or class of DAA is characterized by specific resistance profiles. The likelihood that a DAA will select for and allow outgrowth of viral populations carrying RASs depends on the DAA's genetic barrier to resistance (the number and type of mutations needed to generate an amino acid substitution that confers resistance), the viral fitness (replicative capacity) of the resistant variant, and viral genotypes and subtypes [7, 8] . The prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] .", "The prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] . However, apart from Brazil and Argentina, this issue has not been fully addressed in other South American countries yet [9, [17] [18] [19] . The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] .", "The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] . In this study, we explored the presence of resistance variants to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients chronically infected with hepatitis C. Here, we aimed to contribute to the knowledge of the circulation of HCV resistant variants in the South American region. Samples.", "Samples. Serum samples were obtained from 31 patients with serological markers for HCV, which were recruited between 2015 and 2017 at the Gastroenterology Clinic from Hospital de Clínicas, Montevideo, Uruguay. HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA).", "HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA). Patients selected for this study were both chronically infected with HCV genotype 1 and DAA treatment-naïve at the time of blood extraction. Written informed consent was obtained from all patients.", "Written informed consent was obtained from all patients. The studies have been performed according to the World Medical Association Declaration of Helsinki and approved by the appropriate institutional board (Hospital de Clínicas ethical committee). 2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification.", "2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification. Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol.", "Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol. The viral RNA was heated at 65°C for 5 min and used as a template for a reverse transcription reaction. The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA).", "The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA). The reverse transcription was performed at 42°C for 50 min, and then the reverse transcriptase enzyme was inactivated at 70°C for 15 min. PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously described [10] .", "PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously described [10] . Amplicons were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Science, Buckinghamshire, UK) according to the manufacturer's protocol. 2.3. NS5A and NS5B Sequencing.", "2.3. NS5A and NS5B Sequencing. The purified product was then sequenced using the same sets of primers used for PCR amplification. Bidirectional Sanger sequencing was performed by Macrogen Korea (\n\n2.4. NS5A and NS5B Genotype Determination.", "NS5A and NS5B Genotype Determination. HCV NS5A and NS5B consensus sequences obtained from Uruguayan patients were aligned with sequences from HCV representing all genotypes and main subtypes isolated in different geographic regions of the world. These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] .", "These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] . For strains included in these studies, see Supplementary Material Table S1 . Sequences were aligned using the CLUSTAL W software [23] .", "Sequences were aligned using the CLUSTAL W software [23] . Once aligned, the best evolutionary model that described our sequence data was assessed using ModelGenerator program [24] . Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] .", "Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] . For NS5A, 953 nucleotides (positions 6367 to 7319, relative to HCV 1a reference strain, H77 NC_004102) were included in the phylogenetic analysis, whereas for NS5B, only 361 nucleotides corresponding to the Okamoto region (positions 8265 to 8625, relative to strain H77 NC_004102) were included. As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates).", "As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates). For NS5A 1a Uruguayan sequences (n = 20), a second alignment and maximum likelihood phylogenetic tree was generated in order to analyze HCV evolutionary relationships between Uruguayan, Brazilian, and worldwide strains. For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2.", "For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2. 2.5. NS5A and NS5B Sequence Analysis.", "2.5. NS5A and NS5B Sequence Analysis. In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide.", "In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide. For this purpose, NS5A gene sequences corresponding to subtypes 1a (n = 160) and 1b (n = 88) were retrieved from Los Alamos HCV sequence database and from the NIAID ViPR [21, 22] . Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively.", "Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively. Using Seqman program, implemented in DNAStar 5.01 package (DNASTAR, Madison, USA), a world consensus nucleotide sequences were generated for each gene and subtype. Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated.", "Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated. The amino acid sequences obtained were compared in order to explore the presence of RASs as well as the presence of polymorphisms at a RAS position (RAPs) in Uruguayan HCV strains. RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] .", "RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] . To study the genetic variability of NS5A and NS5B regions of HCV strains circulating in Uruguayan patients, sequences of these regions (accession numbers MH070029-MH070090) were aligned with corresponding sequences from 59 HCV strains isolated elsewhere, representing all genotypes and main subtypes (for strains included in these analyses, see Supplementary Material Table S1 ). Therefore, maximum likelihood phylogenetic trees were constructed.", "Therefore, maximum likelihood phylogenetic trees were constructed. The results of these studies are shown in Figure 1 All strains in the phylogenies were assigned according to their genotype, and each cluster was supported by very high bootstrap values for both analyzed regions. Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b.", "Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b. The results of NS5A (Figure 1 (a)) and NS5B (Figure 1 \n\nGenotype 1b phylogenetic analyses were concordant for both genomic regions in all 31 sequences, suggesting no recombination events between these regions. To further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ).", "To further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ). As was previously described, two distinct 1a clades (clades 1 and 2) were observed. Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] .", "Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] . Whereas NS5A Uruguayan strains (in red) did not cluster in a particular clade, rather, they grouped dispersedly within all major world clades. With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence).", "With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence). AA substitutions at positions previously found to be potentially associated with resistance to NS5A inhibitors, as well as polymorphisms at a RAS position, were identified. These results are summarized in Table 1 .", "These results are summarized in Table 1 . RASs to NS5A inhibitors (L31M and L31V) were identified in 2 strains out of 25 (8%) fully sequenced samples. RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q.", "RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q. Although these substitutions were not reported as resistant, some changes at these positions were previously described as RASs in subtype 1a, namely H58D and K24R [27, 28] . Finally, substitution E62D was found in one subtype 1a strain.", "Finally, substitution E62D was found in one subtype 1a strain. This change is considered as a secondary substitution because, although it does not confer resistance by itself, when combined with a known RAS it does. In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] .", "In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] . In addition, several polymorphisms that have not been previously reported to be associated with a resistant phenotype were also detected (see Supplementary Material Table S3 ). In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences.", "In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences. Almost full-length AA sequences were obtained in 26 out of 31 analyzed strains. 23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein.", "23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein. This issue limited our studies, since many of the described RASs are observed as of residue 553. Importantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%).", "Importantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%). C451R was found in two isolates while A421V was found in only one. In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed.", "In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed. Finally, we found two RAPs: A421V (in 2 subtype 1b strains) and A553G (in 1 subtype 1a strain). Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] .", "Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] . In position 553, the substitution reported as resistant was A553T [8] . As was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ).", "As was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ). The advent of DAAs therapies constitutes one of the major breakthroughs in HCV infected patients management. However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems.", "However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems. The main limiting factors for worldwide access to DAAs in our region concern the high cost, the inadequate management of public healthcare systems, the limited access of low-income or uninsured populations to healthcare providers, and the lack of accurate epidemiological information [20, [30] [31] [32] . In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases.", "In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases. Despite the high rates of viral response achieved with DAA-based treatments, still 1 to10% of the patients fails to eliminate infection, and in these cases, baseline and emergent resistance variants turn out to be key factors contributing to treatment failure [5, 17, 33] . Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating.", "Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating. These facts could compromise the effectiveness of these new therapies in our country. We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] .", "We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] . The present study describes the prevalence of baseline NS5A and NS5B RASs in HCV genotype 1 infected DAA-naïve patients in a Uruguayan cohort. The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] .", "The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] . However, NS5A sequences from South America are poorly analyzed yet [9, 44] . Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] .", "Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] . Importantly, the prevalence and type of baseline NS5A RASs varies slightly by geographic regions. For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] .", "For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] . For this reason, we believe that there is a need to contribute data from our region, for which we still do not have enough information, apart from Brazil [9, 44] . The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ).", "The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ). L31M substitution confers resistance to daclatasvir (DCV), ledipasvir (LDV), and elbasvir (EBV) in both 1a and 1b subtypes [5, 6, 8, 28, 47, 48] , whereas substitution L31V does it to DCV in subtypes 1a and 1b, to LDV in subtype 1b, and to EBV in subtype 1a [5, 6, 28] . Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] .", "Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] . The substitutions H58P and K24Q found in two patients are considered as resistance-associated polymorphisms (RAPs). The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] .", "The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] . The substitution H58P was found as a baseline RAP in relapsers to LDV (HARVONI prescription, media/files/pdfs/medicines/liver-disease/harvoni/harvoni_pi. pdf?la=en).", "pdf?la=en). However, it is sometimes regarded as a RAS [10, 51] , despite conferring only 1.2 fold change in resistance in in vitro studies using the 1a replicon system [39] . We did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] .", "We did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] . The amino acid substitution E62H was found in one Uruguayan patient. Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] .", "Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] . The presence of baseline NS5A RASs impacts treatment outcome in some patient groups by affecting SVR rates. The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use.", "The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use. Regarding NS5B gene, global analysis (with the exception of South America [17, 19] ) revealed that NS5B DAA resistance substitutions are infrequent [14] . Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%).", "Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%). Substitutions found in this work were A421V and S556G associated in subtype 1a with resistance to BCV and dasabuvir (DSV), respectively [8, 28, 29, 54, 55] , and Q556R associated with resistance to DSV both in genotype 1a and 1b [12, 28] . Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV.", "Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV. In these cases, it appeared in combination with G558R (Trial Coral I-Cohort 2: RAPs in positions 421 and 553 (A421V in two subtype 1b isolates and A553G in one subtype 1b isolate) were also found. Although A421V has been associated with resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] .", "Although A421V has been associated with resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] . In position 553, the substitutions reported as resistant are A553T in subtype 1a [8] and A553V in subtype 1b [54] , conferring resistance to DSV. In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ).", "In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ). Nevertheless, substitution A421V was found in Brazil [17] , Argentina [19] , and Uruguay. The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] .", "The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] . Our findings further confirm and complement previous studies which evidenced a low prevalence of this substitution in vivo, probably due to its low replicative fitness [14, 18, 57] . Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] .", "Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] . These results show both diversity in the baseline polymorphisms found in different Latin American countries and in the evolutionary relationships of Uruguayan isolates ( Figure 2 ). This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host.", "This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host. It is worth mentioning that we live in a vast continent inhabited by populations with different genotypic characteristics that might, depending on the situation, require different approaches to treatment. Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] .", "Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] . Altogether, we believe that it could be important to carry out studies throughout the South American region in order to establish the prevalence of RASs in NS5A and NS5B in different countries. In fact, this will aid in understanding that not every treatment regimen might be adequate for every patient and country.", "In fact, this will aid in understanding that not every treatment regimen might be adequate for every patient and country. The data we presented here might guide not only physicians in making therapeutic decisions but also public health authorities in approving more diverse treatment combinations. These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population.", "These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population. To our knowledge, the present study revealed for the first time the presence of RASs in the NS5A and NS5B regions of HCV genotype 1 Uruguayan strains from patients who have not been previously treated with DAAs and is one of the few South American countries to report on this matter. It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure.", "It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure. However, individualized DAA therapy based on baseline resistance analysis may be beneficial for optimizing treatment efficacy in patients with HCV genotype 1 infection and risk factors for treatment failure. Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions.", "Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions. The data used to support the findings of this study are included within the article. The authors declare that they have no conflicts of interest. Fabián Aldunate and Natalia Echeverría contributed equally to this work.", "Fabián Aldunate and Natalia Echeverría contributed equally to this work. Supplementary Material Table S1 : hepatitis C Virus NS5A and NS5B sequences used as representatives of each genotype to perform the phylogenetic analysis. Their corresponding genotype, country of isolation, and GenBank accession number are indicated.", "Their corresponding genotype, country of isolation, and GenBank accession number are indicated. Supplementary Material Table S2 : hepatitis C Virus NS5A subtype 1a sequences used to reveal evolutionary relationships between Uruguayan strains and others isolated elsewhere. Their corresponding country of isolation and GenBank accession number are indicated.", "Their corresponding country of isolation and GenBank accession number are indicated. Supplementary Material Table S3 : amino acid substitutions in NS5A protein not previously associated with resistance to NS5A inhibitors. Supplementary Material Table S4 : amino acid substitutions in NS5B protein not previously associated with resistance to polymerase inhibitors. (Supplementary Materials)" ]

[ "BACKGROUND: Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria.", "This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. RESULTS: A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria.", "Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that modifications in the E2 protein coding sequence were stably maintained. A single amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence.", "A single amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence. CONCLUSIONS: These results show that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses. Text: Bacterial artificial chromosomes (BACs) are ideally suited for the stable maintenance of large DNA sequences derived from viral genomes [1] .", "Text: Bacterial artificial chromosomes (BACs) are ideally suited for the stable maintenance of large DNA sequences derived from viral genomes [1] . A considerable number of BAC systems have been established for large DNA viruses; in particular many different herpesvirus genomes have been cloned into BACs (for review see [2] ). The first BAC systems using RNA virus cDNAs were described for coronaviruses [3] [4] [5] [6] and recently the first BAC containing a full-length cDNA for a negative-stranded RNA virus was described [7] .", "The first BAC systems using RNA virus cDNAs were described for coronaviruses [3] [4] [5] [6] and recently the first BAC containing a full-length cDNA for a negative-stranded RNA virus was described [7] . Similarly, cDNAs corresponding to the full-length genomes of members of the Flaviviridae family (Japanese encephalitis virus [8] and Dengue virus [9] ) have been inserted into BACs. BACs containing full-length cDNAs of pestiviruses (also within the Flaviviridae), including bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) have recently been established [10, 11] .", "BACs containing full-length cDNAs of pestiviruses (also within the Flaviviridae), including bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) have recently been established [10, 11] . Infectious pestiviruses can be rescued using RNA transcripts derived from these BACs. The pestiviruses have single stranded positive sense RNA genomes, about 12.3 kb in length, which includes a single long open reading frame, encoding a large polyprotein, flanked by 5′ and 3′ untranslated regions (UTRs) that are critical for autonomous replication of the genome [12, 13] .", "The pestiviruses have single stranded positive sense RNA genomes, about 12.3 kb in length, which includes a single long open reading frame, encoding a large polyprotein, flanked by 5′ and 3′ untranslated regions (UTRs) that are critical for autonomous replication of the genome [12, 13] . The polyprotein is cleaved by cellular and viral proteases into four structural proteins (nucleocapsid protein C, envelope glycoproteins E rns , E1 and E2) and eight nonstructural proteins (N pro , p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). The availability of genetically defined and stable pestivirus BACs facilitates the functional study of viral proteins or RNA structures and also the development of new marker vaccine candidates.", "The availability of genetically defined and stable pestivirus BACs facilitates the functional study of viral proteins or RNA structures and also the development of new marker vaccine candidates. Several CSFV vaccines with marker properties based on chimeric pestiviruses have been developed over the years [14] . In particular, chimeric pestiviruses with substitution of the entire E2 protein have been described [15] [16] [17] but also mutants with more subtle modifications, such as the modification of the important TAV-epitope [18] within the CSFV-E2 protein [19, 20] are promising marker vaccine candidates.", "In particular, chimeric pestiviruses with substitution of the entire E2 protein have been described [15] [16] [17] but also mutants with more subtle modifications, such as the modification of the important TAV-epitope [18] within the CSFV-E2 protein [19, 20] are promising marker vaccine candidates. Manipulation of BACs using traditional cloning procedures can be difficult (e.g. because of a lack of convenient restriction enzyme sites) and thus a range of methodologies that apply bacterial genetics, including homologous recombination (e.g.", "because of a lack of convenient restriction enzyme sites) and thus a range of methodologies that apply bacterial genetics, including homologous recombination (e.g. Red/ET homologous recombineering) within the E. coli host, have been developed (for review, see [21] ). The use of homologous recombination allows site-directed mutagenesis of BACs [22] and, by employing a counterselection scheme, specific modifications can be obtained without leaving residual \"foreign\" sequences [23] .", "The use of homologous recombination allows site-directed mutagenesis of BACs [22] and, by employing a counterselection scheme, specific modifications can be obtained without leaving residual \"foreign\" sequences [23] . The main advantage of this method is that there are no target limitations (e.g. based on size or location) and no need for suitable restriction sites.", "based on size or location) and no need for suitable restriction sites. The integration of the modified sequence is performed in vivo (within E. coli) thereby potentially being more accurate than in vitro approaches like PCR-based methods. Although in vitro cloning approaches based on the use of high-fidelity polymerases for PCR amplification have significantly improved in recent years, the use of in vivo approaches should allow a more accurate method of mutagenesis due to the use of the cells own high-fidelity replication system which includes proof reading.", "Although in vitro cloning approaches based on the use of high-fidelity polymerases for PCR amplification have significantly improved in recent years, the use of in vivo approaches should allow a more accurate method of mutagenesis due to the use of the cells own high-fidelity replication system which includes proof reading. Whereas BAC recombination has been commonly used for modifying DNA viruses, there are only very few reports about the use of this technology for RNA viruses [7, 24, 25] . Here, a generally applicable strategy for the manipulation and rescue of chimeric pestiviruses from BACs is described as a model, and the flexibility of this approach is demonstrated by generating different modifications in the viral cDNA of the new CSFV-BAC, pBeloR26, derived from the modified live vaccine strain \"C-strain Riems\".", "Here, a generally applicable strategy for the manipulation and rescue of chimeric pestiviruses from BACs is described as a model, and the flexibility of this approach is demonstrated by generating different modifications in the viral cDNA of the new CSFV-BAC, pBeloR26, derived from the modified live vaccine strain \"C-strain Riems\". The targeted recombination-mediated mutagenesis described here includes the substitution of the 9 amino acid (aa) linear TAV-epitope (TAVSPTTLR) present in the E2 protein with the corresponding region (TTVSTSTLA) of a heterologous pestivirus (border disease virus, BDV, strain \"Gifhorn\") and also the replacement of the entire CSFV E2 protein coding region with the whole E2 coding region from the same BDV, to generate marked vaccine viruses that can be discriminated using specific anti-E2 monoclonal antibodies. The genetic stabilities of both the BAC constructs (within E. coli) and the rescued viruses have also been assessed.", "The genetic stabilities of both the BAC constructs (within E. coli) and the rescued viruses have also been assessed. Porcine kidney (PK15) and sheep fetal thymoid (SFT-R) cells were grown at 37°C (with 5% (v/v) CO 2 ) in Dulbecco's minimal essential medium (DMEM) supplemented with 5% (v/v) pestivirus-free fetal calf serum. Virus from a bait containing the modified live vaccine CSFV \"C-strain Riems\" (Riemser Arzneimittel AG, Germany) was propagated once in PK15 cells and termed vRiemser.", "Virus from a bait containing the modified live vaccine CSFV \"C-strain Riems\" (Riemser Arzneimittel AG, Germany) was propagated once in PK15 cells and termed vRiemser. RNA obtained from BDV strain \"Gifhorn\" [26] was used for amplification of the Gifhorn E2-coding sequence. Oligonucleotide primers used are listed in Additional file 1: Table S1 .", "Oligonucleotide primers used are listed in Additional file 1: Table S1 . The BAC construct, pBeloR26, was constructed using the long RT-PCR method as previously described [11] using RNA derived from the \"C-strain Riems\". Briefly, full-length viral cDNAs flanked by NotI sites were amplified by long RT-PCR using primers 5′Cstrain_T7_Not1 (which includes a T7 promotor for in vitro transcription, a NotI site and a region corresponding to the first 44 nt of the genome) and 3′CSFV_Not1 (that contains a NotI site and sequence complementary to the 3′-terminal 35 nt of the genome that are conserved among many CSFVs including the Cstrain).", "Briefly, full-length viral cDNAs flanked by NotI sites were amplified by long RT-PCR using primers 5′Cstrain_T7_Not1 (which includes a T7 promotor for in vitro transcription, a NotI site and a region corresponding to the first 44 nt of the genome) and 3′CSFV_Not1 (that contains a NotI site and sequence complementary to the 3′-terminal 35 nt of the genome that are conserved among many CSFVs including the Cstrain). The product (ca. 12.3 kbp) was digested with NotI and inserted into similarly digested pBeloBAC11 (New England Biolabs, GenBank accession U51113).", "12.3 kbp) was digested with NotI and inserted into similarly digested pBeloBAC11 (New England Biolabs, GenBank accession U51113). All BACs were modified and maintained in E. coli DH10B cells (Invitrogen) grown at 37°C in LB medium containing chloramphenicol (Cam, 15 μg/ml). The electroporation of bacteria was performed in 0.1 cm cuvettes using 1 pulse at 1800 V, 25 μF and 200 Ω in a Gene Pulser Xcell (Bio-Rad).", "The electroporation of bacteria was performed in 0.1 cm cuvettes using 1 pulse at 1800 V, 25 μF and 200 Ω in a Gene Pulser Xcell (Bio-Rad). BACs to be used as templates for long PCR or for screening by restriction enzyme digestion were purified from 4 ml overnight cultures of E. coli DH10B using the ZR BAC DNA Miniprep Kit (Zymo Research). BACs required for direct genome sequencing were purified from 500 ml cultures using the Large-construct kit (Qiagen).", "BACs required for direct genome sequencing were purified from 500 ml cultures using the Large-construct kit (Qiagen). Modifications to the full-length CSFV cDNA were accomplished in E. coli DH10B (streptomycin resistant, Strep R ) using the Counter Selection BAC Modification Kit (Gene Bridges, Heidelberg, Germany). The Red/ET recombination involved three steps (i-iii).", "The Red/ET recombination involved three steps (i-iii). Step i) the temperature-sensitive pRedET expression plasmid (Gene Bridges) was introduced into electroporationcompetent E.coli DH10B cells containing the parental BAC (phenotype Cam R , Strep R ). The pRedET expresses the phage lambda proteins redα, redβ and redγ, under control of the arabinose-inducible pBAD promoter, allowing homologous recombination to occur.", "The pRedET expresses the phage lambda proteins redα, redβ and redγ, under control of the arabinose-inducible pBAD promoter, allowing homologous recombination to occur. Immediately after electroporation, pre-warmed LB medium without antibiotics (1 ml) was added to the cells which were then incubated at 30°C for 1 hour, prior to spreading onto agar plates containing Cam (15 μg/ml) and tetracycline (Tet) (3 μg/ml) and then incubated at 30°C overnight to maintain the pRedET. The presence of the pRedET plasmid (conferring Tet R ) was verified by visual inspection of BAC-DNA preparations from the Cam R /Tet R colonies using agarose gel electrophoresis.", "The presence of the pRedET plasmid (conferring Tet R ) was verified by visual inspection of BAC-DNA preparations from the Cam R /Tet R colonies using agarose gel electrophoresis. Step ii) counter-selection marker cassettes with an extra NotI site for screening purposes (rpsL-neo, 1325 bp) were amplified by PCR using primers with 30 nt or 50 nt extensions that were homologous to the target site in the BAC using the rpsL-neo plasmid (Gene Bridges) as template and the Phusion hot start II HF DNA polymerase (Thermo Scientific) with cycling conditions as follows: 98°C for 30s, followed by 35 cycles of 98°C for 10s, 60°C for 20s, 72°C for 60s, and 1 cycle at 72°C for 4 min. The PCR products (ca.", "The PCR products (ca. 1400 bp) were isolated on 1% (w/v) TBE agarose gels and purified using a GeneJET gel extraction kit (Thermo Scientific). Samples (30 μl), from an E. coli culture containing pRedET and the parental BAC grown overnight at 30°C in LB media (Cam, Tet), were used to inoculate 1.4 ml of fresh LB media with the same antibiotics to obtain exponentially growing bacteria at 30°C.", "Samples (30 μl), from an E. coli culture containing pRedET and the parental BAC grown overnight at 30°C in LB media (Cam, Tet), were used to inoculate 1.4 ml of fresh LB media with the same antibiotics to obtain exponentially growing bacteria at 30°C. Red/ET recombination proteins were induced by adding 50 μl of 10% (w/v) L-arabinose (Sigma). The PCR product (200 ng) containing the rpsL-neo cassette was introduced into these bacteria using electroporation (as above).", "The PCR product (200 ng) containing the rpsL-neo cassette was introduced into these bacteria using electroporation (as above). Following electroporation, the cells were grown at 37°C for 70 min (to allow recombination) and then selected on plates containing Cam (15 μg/ml), Tet (3 μg/ml) and kanamycin (Kan, 15 μg/ml) overnight at 30°C to maintain the pRedET. Note, the rpsL cassette confers Streptomycin sensitivity (Strep S ) onto the resistant DH10B strain and the neo confers Kanamycin resistance (Kan R ).", "Note, the rpsL cassette confers Streptomycin sensitivity (Strep S ) onto the resistant DH10B strain and the neo confers Kanamycin resistance (Kan R ). The correct phenotype (Cam R , Kan R , Tet R , Strep S ) of the resulting colonies was confirmed by streaking the colonies onto plates containing Cam (15 μg/ml), Tet (3 μg/ml) and Kan (15 μg/ml) and grown at 30°C. Importantly, for the third step, the replacement of the rpsL-neo cassette (using counter-selection), the selected colonies were also streaked onto plates containing Cam (15 μg/ml) plus Strep (50 μg/ml) and shown to be Strep S indicating incorporation of a functional rpsL gene.", "Importantly, for the third step, the replacement of the rpsL-neo cassette (using counter-selection), the selected colonies were also streaked onto plates containing Cam (15 μg/ml) plus Strep (50 μg/ml) and shown to be Strep S indicating incorporation of a functional rpsL gene. The structures of the intermediate BACs were verified by restriction enzyme analysis and sequencing around the inserts. Step iii) the replacement of the rpsL-neo selection cassettes from the intermediate constructs using linear DNA fragments was achieved through counter-selection and Red/ET recombination.", "Step iii) the replacement of the rpsL-neo selection cassettes from the intermediate constructs using linear DNA fragments was achieved through counter-selection and Red/ET recombination. Again, the homologous sequences at the ends of the DNA fragment were used for Red/ET mediated recombination events to replace the rpsL-neo cassette with the sequence of interest. Counterselection against the rpsL-neo cassette (phenotype Cam R , Kan R , Tet R , Strep S ) was employed using media containing Cam (15 μg/ml) and Strep (50 μg/ml) to isolate the required derivatives (phenotype Cam R and Strep R ).", "Counterselection against the rpsL-neo cassette (phenotype Cam R , Kan R , Tet R , Strep S ) was employed using media containing Cam (15 μg/ml) and Strep (50 μg/ml) to isolate the required derivatives (phenotype Cam R and Strep R ). Initially, the intermediate construct, pBeloR26_E2rpsLneo ( Figure 1 ), was generated using Red/ET recombination by insertion of the rpsL-neo cassette with an extra NotI site for screening purposes which was amplified using primers Criems-TAVfor and Criems-TAVrev (Additional file 1: Table S1 ) in place of the TAVSPTTLR coding sequence (27 nt) . Secondly, the rpsL-neo cassette in this intermediate construct was then replaced using counter-selection Red/ ET recombination using a single-stranded oligonucleotide, Riems_TAV_Gifhorn (Additional file 1: Table S1 ) with the same homology arms as used for the rpsL-neo cassette, to introduce the coding sequence for the BDV \"Gifhorn\" epitope sequence (TTVSTSTLA).", "Secondly, the rpsL-neo cassette in this intermediate construct was then replaced using counter-selection Red/ ET recombination using a single-stranded oligonucleotide, Riems_TAV_Gifhorn (Additional file 1: Table S1 ) with the same homology arms as used for the rpsL-neo cassette, to introduce the coding sequence for the BDV \"Gifhorn\" epitope sequence (TTVSTSTLA). The resulting construct was named pBeloR26_TAV (Figure 1 ). The initial intermediate construct (with rpsL-neo) was then used to produce the pBeloR26_E2gif construct ( Figure 1 ).", "The initial intermediate construct (with rpsL-neo) was then used to produce the pBeloR26_E2gif construct ( Figure 1 ). For this, the E2 coding sequence was amplified from cDNA prepared from BDV \"Gifhorn\" RNA using two different primer pairs, one set with 50 nt homology arms (Criems_E2_gifFlong/Criems_ E2_gifRlong) and another with 30 nt homologous sequences (Criems_E2_gifF/Criems_E2_gifR). For generation of BACs with substitution of the entire E2 coding sequences, PCR products consisting of the sequence of interest flanked with homology arms identical to the target area were generated by PCR (as for the rpsLneo cassette).", "For generation of BACs with substitution of the entire E2 coding sequences, PCR products consisting of the sequence of interest flanked with homology arms identical to the target area were generated by PCR (as for the rpsLneo cassette). For making constructs with substitution of shorter sequences (e.g. the TAV-epitope), the recombination was achieved using synthetic single stranded oligonucleotides rather than PCR products.", "the TAV-epitope), the recombination was achieved using synthetic single stranded oligonucleotides rather than PCR products. Pre-heating of single stranded oligonucleotides at 95°C for 2 min followed by snap-freezing, prior to electroporation, empirically showed the best results. In each case, the DNA molecules were introduced into E. coli containing the BAC derivatives including the rpsL-neo cassettes together with the pRedET plasmid by electroporation as described above.", "In each case, the DNA molecules were introduced into E. coli containing the BAC derivatives including the rpsL-neo cassettes together with the pRedET plasmid by electroporation as described above. The structures of the modified BACs were verified by restriction enzyme analysis and subsequent full-genome sequencing (see below). BAC DNA (1 μg) was linearized with NotI or 1 μl BAC DNA was used as template for long PCR amplification using primers 5′C-strain_T7_Not1 and 3′CSFV (Additional file 1: Table S1 ).", "BAC DNA (1 μg) was linearized with NotI or 1 μl BAC DNA was used as template for long PCR amplification using primers 5′C-strain_T7_Not1 and 3′CSFV (Additional file 1: Table S1 ). Linearized BACs or PCR products were purified with the GeneJet PCR purification kit (Thermo Scientific) and transcribed in vitro using a Megascript T7 kit (Invitrogen). Viruses were rescued from RNA transcripts (1 to 5 μg) by electroporation of porcine (PK15) or ovine (SFT-R) cells essentially as described previously [24] .", "Viruses were rescued from RNA transcripts (1 to 5 μg) by electroporation of porcine (PK15) or ovine (SFT-R) cells essentially as described previously [24] . Cells were analysed using immunofluorescence microscopy (typically after 3 days) for the expression of NS3 and E2 proteins using specific monoclonal antibodies (mAbs), these were anti-NS3 (WB103/105, pan-pestivirus), anti-CSFV E2 (WH211, WH303, both CSFV specific) and anti-BDV E2 (WB166, BVDV/BDV specific) (AHVLA Scientific, United Kingdom) together with Alexa 488 conjugated goat antimouse IgG antibody (Molecular Probes, Invitrogen). The nuclei of cells were visualized using DAPI (Vector Laboratories) and images were recorded using a BX63 fluorescence microscope (Olympus).", "The nuclei of cells were visualized using DAPI (Vector Laboratories) and images were recorded using a BX63 fluorescence microscope (Olympus). For peroxidase staining, cells were fixed and stained for the presence of pestivirus antigens using biotinylated pig anti-CSFV/BVDV polyclonal IgG followed by avidin-conjugated horseradish peroxidase (eBioscience) as previously described [27] . The same staining procedure was also performed using the anti-E2 mAbs.", "The same staining procedure was also performed using the anti-E2 mAbs. Samples containing virus-positive cells were passaged onto new cells. Virus growth curves were generated as previously described [24] . Briefly, PK15 or SFT-R cells were infected at a multiplicity of infection (MOI) of 0.1 pfu/cell and grown for three days.", "Briefly, PK15 or SFT-R cells were infected at a multiplicity of infection (MOI) of 0.1 pfu/cell and grown for three days. BAC DNAs (5 μg), purified using the Large-construct kit (Qiagen), or PCR products (1 μg) amplified from viral cDNA or from BACs using the long PCR method (as above) were consensus sequenced using a 454 FLX (Roche) or an Ion PGM (Life Technologies). Both Newbler (Roche) and the bwa.bwasw alignment algorithm [28] were used for mapping the reads to the expected sequence.", "Both Newbler (Roche) and the bwa.bwasw alignment algorithm [28] were used for mapping the reads to the expected sequence. A combination of Samtools [29] and LoFreq SNV-caller [30] was used for downstream single nucleotide variant (SNV) analysis. Finally, clone consensus sequences were aligned using MAFFT in the Geneious software platform (Biomatters).", "Finally, clone consensus sequences were aligned using MAFFT in the Geneious software platform (Biomatters). Generation of a BAC containing full-length cDNA corresponding to the modified live vaccine \"C-strain Riems\"\n\nBACs containing the full-length cDNA corresponding to the parental vRiemser (\"C-strain Riems\") were constructed according to the method described previously for the \"Paderborn\" strain of CSFV [11] . BACs containing the complete CSFV cDNAs were identified by restriction Figure 1 Schematic representation of the CSFV genome organization and the BACs constructed and used in this study.", "BACs containing the complete CSFV cDNAs were identified by restriction Figure 1 Schematic representation of the CSFV genome organization and the BACs constructed and used in this study. Nucleotide (nt) and amino acid (aa) positions within R26 for the 5′ and 3′ termini together with the translational start and stop codons of the polyprotein coding region plus cleavage sites used to make the individual proteins (N pro , C, E rns , E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) are indicated. Insertion of the rpsL-neo in place of the TAV-epitope within CSFV E2 for the intermediate construct (R26_rpsLneo) and the subsequent replacement with the TTVSTSTLA sequence (R26_TAV) and the complete substitution of the E2 sequence (R26_E2gif) are shown.", "Insertion of the rpsL-neo in place of the TAV-epitope within CSFV E2 for the intermediate construct (R26_rpsLneo) and the subsequent replacement with the TTVSTSTLA sequence (R26_TAV) and the complete substitution of the E2 sequence (R26_E2gif) are shown. Names of BAC constructs begin with \"pBelo\" and rescued viruses with \"v\" (e.g. pBeloR26 and vR26).", "pBeloR26 and vR26). Cell culture passage no. of virus is indicated with \"/P\" (e.g. vR26/P-4). digest analysis and following linearization by NotI, RNA transcripts were produced and electroporated into PK15 cells.", "digest analysis and following linearization by NotI, RNA transcripts were produced and electroporated into PK15 cells. This screening resulted in the identification of a BAC containing a cDNA insert of 12316 nt, pBeloR26 (Figure 1) , which yielded infectious virus, termed vR26, that could be propagated in SFT-R cells (Figure 2 , upper panels) and in PK15 cells (Figure 3 ). The rescued vR26 displayed higher growth rate at the early stage (about 10fold difference in virus yield at 24 h) compared to the parental vaccine virus, but after 48 hours similar virus titres were obtained (Figure 3 ).", "The rescued vR26 displayed higher growth rate at the early stage (about 10fold difference in virus yield at 24 h) compared to the parental vaccine virus, but after 48 hours similar virus titres were obtained (Figure 3 ). Full-genome sequencing of the cloned BAC template, pBeloR26, revealed a number of differences throughout the genome when compared to the full-length consensus sequence of the cDNA used for the cloning procedure (see Table 1 ). These differences are non-representative variants within the cDNA.", "These differences are non-representative variants within the cDNA. Overall, the BAC sequence differed from the cDNA sequence in 18 positions, 9 of these lead to predicted amino acid substitutions within the polyprotein; one in each of N pro , E rns , E1, E2 and NS3 and four amino acid substitutions in NS5B (Table 1) . When compared to the published reference sequence (GenBank accession AY259122.1), the pBeloR26 BAC sequence differed at an additional 11 positions, 1 of these lead to a predicted amino acid substitution and there was one large insertion (27 nt) in the hypervariable region of the 3′-UTR (Additional file 2: Table S2 ).", "When compared to the published reference sequence (GenBank accession AY259122.1), the pBeloR26 BAC sequence differed at an additional 11 positions, 1 of these lead to a predicted amino acid substitution and there was one large insertion (27 nt) in the hypervariable region of the 3′-UTR (Additional file 2: Table S2 ). To determine the utility of the targeted recombinationmediated mutagenesis system for pestiviruses, two different modifications of the E2 protein coding sequence within pBeloR26 were generated using the Red/ET recombination methodology. Initially, the sequence encoding the linear TAV-epitope (TAVSPTTLR) within the CSFV-E2 was substituted with the sequence encoding the corresponding region (encoding TTVSTSTLA) from the BDV strain \"Gifhorn\" as described in the Materials and Methods section.", "Initially, the sequence encoding the linear TAV-epitope (TAVSPTTLR) within the CSFV-E2 was substituted with the sequence encoding the corresponding region (encoding TTVSTSTLA) from the BDV strain \"Gifhorn\" as described in the Materials and Methods section. More than 90% of the colonies obtained using this procedure contained the required BAC\n\nAnti-CSFV E2 (WH211) Figure 2 Antibody reaction patterns of pestivirus infected cells. SFT-R cells were infected with vR26 and its two derivatives vR26_E2gif and vR26_TAV plus vGifhorn [26] .", "SFT-R cells were infected with vR26 and its two derivatives vR26_E2gif and vR26_TAV plus vGifhorn [26] . After 72 h, the cells were fixed and stained with monoclonal antibodies against the NS3 protein (WB103/105, left column), the CSFV E2 protein (WH303 and WH211, middle columns) and the BDV E2 protein (WB166, right column) as indicated and viewed using a fluorescence microscope. structure as determined by NotI digestions.", "structure as determined by NotI digestions. The complete genome sequences of the CSFV cDNA within two selected BACs, designated pBeloR26_TAV have been verified (data not shown). In addition, the complete coding sequence (1119 nt) for the CSFV-E2 protein was substituted by the corresponding sequence from BDV \"Gifhorn\".", "In addition, the complete coding sequence (1119 nt) for the CSFV-E2 protein was substituted by the corresponding sequence from BDV \"Gifhorn\". Again more than 90% of the colonies obtained contained the required BAC and the same proportion of correctly recombined BACs was obtained using either 30 nt or 50 nt homology arms. The chimeric BAC was designated, pBeloR26_E2gif and the complete virus genome sequence (cDNA) was verified (data not shown).", "The chimeric BAC was designated, pBeloR26_E2gif and the complete virus genome sequence (cDNA) was verified (data not shown). After electroporation with RNA transcripts derived from either pBeloR26_TAV or pBeloR26_E2gif a large number of CSFV NS3-positive cells could be observed (data not shown) and chimeric virus stocks, termed vR26_TAV and vR26_E2gif, were generated after further passages in cells. Cells infected with these viruses and with the parental vR26 and vGifhorn strains were all stained with mAbs directed against the NS3 protein ( Figure 2 ).", "Cells infected with these viruses and with the parental vR26 and vGifhorn strains were all stained with mAbs directed against the NS3 protein ( Figure 2 ). However, in contrast to the parental vR26 virus, the chimeric viruses rescued from the recombined BACs were not recognized by anti-E2 mAbs specific for the CSFV-E2 proteins ( Figure 2 ) and thus, consistent with their structure, displayed the same antibody reaction pattern as vGifhorn. Two different anti-CSFV E2 mAbs, WH211 and WH303, were used for the staining and the latter has been shown previously to target the TAV-epitope [18] .", "Two different anti-CSFV E2 mAbs, WH211 and WH303, were used for the staining and the latter has been shown previously to target the TAV-epitope [18] . As anticipated, cells infected with either the vGifhorn or with the chimeric vR26_E2gif could be shown to express the \"Gifhorn\" E2 protein using staining with an anti-BDV mAb ( Figure 2 ). The presence of the BDV epitope TTVSTSTLA in vR26_ TAV was insufficient to permit efficient recognition by this anti-BDV mab, although a weak signal was observed in some cells.", "The presence of the BDV epitope TTVSTSTLA in vR26_ TAV was insufficient to permit efficient recognition by this anti-BDV mab, although a weak signal was observed in some cells. The BAC constructs pBeloR26 and pBeloR26_E2gif were analysed for the genetic stability of the cDNA to determine the suitability of the BAC vector for maintaining full-length pestivirus cDNAs. E. coli DH10B cells containing the BACs were passaged 15 times, by overnight growth, and the complete viral cDNAs within the BACs were sequenced after the 1st and the 15th passage.", "E. coli DH10B cells containing the BACs were passaged 15 times, by overnight growth, and the complete viral cDNAs within the BACs were sequenced after the 1st and the 15th passage. No mutations were observed within the 12316 nt virus cDNA sequences after this extensive propagation of the BACs in the bacterial host, indicating a highly stable system for the maintenance of complete pestivirus cDNA sequences. The viruses, vR26 and vR26_E2gif, rescued from their respective BAC constructs, were also tested for their genetic stability within mammalian cells.", "The viruses, vR26 and vR26_E2gif, rescued from their respective BAC constructs, were also tested for their genetic stability within mammalian cells. Linearized BAC DNA was transcribed in vitro and the RNA was electroporated into PK15 cells. Three days after electroporation the cells were stained with the anti-NS3 antibody to detect the presence of replicating virus.", "Three days after electroporation the cells were stained with the anti-NS3 antibody to detect the presence of replicating virus. Samples containing virus positive cells were passaged onto new cells, this process \n\n*Nt position 10665 in vR26/P-12 is reverted from A to G as in the parental cDNA. was repeated for 12 separate passages (each of three days).", "was repeated for 12 separate passages (each of three days). The virus titre (as TCID 50 /ml) was determined for each passage. Passage of the rescued vR26_E2gif chimeric virus in PK15 cells resulted in rapidly decreasing virus titres and was discontinued after the 2nd passage ( Figure 4A ).", "Passage of the rescued vR26_E2gif chimeric virus in PK15 cells resulted in rapidly decreasing virus titres and was discontinued after the 2nd passage ( Figure 4A ). Instead, further passage of this chimeric virus was performed in ovine SFT-R cells (the preferred cell type for BDV) and resulted in much higher titers of the chimeric virus. Virus titers reached more than 10 6 TCID 50 /ml after the 1st passage and remained stable for 12 passages ( Figure 4A ).", "Virus titers reached more than 10 6 TCID 50 /ml after the 1st passage and remained stable for 12 passages ( Figure 4A ). The rescued vR26 was also efficiently propagated on the SFT-R cells but maintained a slightly lower titer than the vR26_E2gif chimeric virus ( Figure 4A ). To check that the viruses retained their antibody reaction properties ( Figure 2 ) after these passages, cells were infected with viruses from the 12th SFT-R cell culture passage (termed vR26/P-12 and vR26_E2gif/P-12) and stained with a polyclonal anti-pestivirus serum and with specific mAbs directed against the CSFV-E2 and BDV-E2 proteins ( Figure 4B ).", "To check that the viruses retained their antibody reaction properties ( Figure 2 ) after these passages, cells were infected with viruses from the 12th SFT-R cell culture passage (termed vR26/P-12 and vR26_E2gif/P-12) and stained with a polyclonal anti-pestivirus serum and with specific mAbs directed against the CSFV-E2 and BDV-E2 proteins ( Figure 4B ). Cells infected with either the vR26/P-12 or the chimeric vR26_E2gif/P-12 were each detected by the polyclonal anti-pestivirus serum as expected. The anti-CSFV-E2 mAb specifically detected cells infected with vR26/P-12 but not cells infected by the chimeric virus containing the BDV-E2 protein (consistent with the results shown in Figure 2 ).", "The anti-CSFV-E2 mAb specifically detected cells infected with vR26/P-12 but not cells infected by the chimeric virus containing the BDV-E2 protein (consistent with the results shown in Figure 2 ). In contrast, the anti-BDV-E2 mAb specifically detected infection by the vR26_E2gif/P-12 and did not recognize cells infected with vR26/P-12. Each result is in accord with the structure of the viruses.", "Each result is in accord with the structure of the viruses. The 4th passage of vR26 (vR26/P-4) displayed a slower growth rate than the virus obtained after 12 passages (see Figure 5A ). It also had a reduced growth rate compared to both the vR26_E2gif/P-4 and vR26_E2gif/P-12.", "It also had a reduced growth rate compared to both the vR26_E2gif/P-4 and vR26_E2gif/P-12. The fulllength sequence of pBeloR26 had revealed ten non-silent mutations compared to the reference sequence (AY25 9122.1) for this virus (Additional file 2: Table S2 ). Any of these mutations could be responsible for the impaired growth acting alone or in concert.", "Any of these mutations could be responsible for the impaired growth acting alone or in concert. For further investigation of this issue, full length cDNAs prepared from vR26/ P-4, vR26/P-12, vR26_E2gif/P-4 and vR26_E2gif/P-12 were deep-sequenced using both the 454 FLX and Ion PGM platforms for comparison and to determine the quasispecies distribution (Additional file 3: Figure S1 and Additional file 4: Figure S2 ). Sequencing data from both platforms revealed that both the vR26/P-12 and vR26_E2gif/P-12 were close to 100% changed at nt position A10665G compared to the BAC clones (resulting in the predicted amino acid substitution D3431G within the NS5B protein, the RNAdependent RNA polymerase, see Figure 5B ).", "Sequencing data from both platforms revealed that both the vR26/P-12 and vR26_E2gif/P-12 were close to 100% changed at nt position A10665G compared to the BAC clones (resulting in the predicted amino acid substitution D3431G within the NS5B protein, the RNAdependent RNA polymerase, see Figure 5B ). This adaptation is a reversion back to the consensus cDNA sequence of the parental vaccine virus, vRiemser (Additional file 2: Table S2 ). Additionally, vR26/P-4 and vR26_E2gif/P-4 already showed evidence for this reversion being present within the population.", "Additionally, vR26/P-4 and vR26_E2gif/P-4 already showed evidence for this reversion being present within the population. For vR26/P-4, the level of reversion was 57%, while for vR26_E2gif/P-4 the extent of change was 73% (see Figure 5B ). In this study, we have established the first BAC containing the full-length cDNA of a CSFV vaccine strain.", "In this study, we have established the first BAC containing the full-length cDNA of a CSFV vaccine strain. The BAC differed from the parental cDNA sequence in 18 positions leading to 9 aa substitutions ( Table 1 ). The method that has been used for the generation of pBeloR26 is based on full genome amplification of cDNA followed by direct cloning to obtain the BACs [11] .", "The method that has been used for the generation of pBeloR26 is based on full genome amplification of cDNA followed by direct cloning to obtain the BACs [11] . This approach results in cDNA clones that reflect the quasispecies composition of the parental viral RNA and thus it is not guaranteed to obtain cDNA clones corresponding to the consensus sequence of the cDNA used. However, it is possible to correct the mutations using the BAC recombination approach if a consensus clone is needed.", "However, it is possible to correct the mutations using the BAC recombination approach if a consensus clone is needed. To demonstrate the utility of the Red/ET mediated recombination method we have generated a series of modified BACs derived from this CSFV full-length cDNA. These include BACs with substitution of the linear TAV-epitope present in the E2 protein and also BACs with substitution of the complete E2 protein with heterologous pestivirus sequences.", "These include BACs with substitution of the linear TAV-epitope present in the E2 protein and also BACs with substitution of the complete E2 protein with heterologous pestivirus sequences. We have also used the same approach for a range of different targeted modifications within CSFV BACs including specific deletions and substitutions in the 5′UTR of CSFV [24] and for insertions of heterologous reporter sequences into CSFV replicons [25] . Using Red/ET recombinationmediated mutagenesis for the targeted design, the work can be expedited and focused, in principal, on any sequence within the viral genome and is not dependent on the use of internal restriction sites.", "Using Red/ET recombinationmediated mutagenesis for the targeted design, the work can be expedited and focused, in principal, on any sequence within the viral genome and is not dependent on the use of internal restriction sites. The results demonstrate that Red/ ET recombination-mediated mutagenesis of pestivirus BAC cDNAs provides a useful tool for advancing the construction of modified pestiviruses. Cells infected with the parental vR26 virus were recognized by the two anti-E2 mAbs (WH211 and WH303) specific for the CSFV-E2 proteins, in contrast cells infected with the modified viruses vR26_TAV and vR26_E2gif, rescued from the recombined BACs, were not detected by these mAbs.", "Cells infected with the parental vR26 virus were recognized by the two anti-E2 mAbs (WH211 and WH303) specific for the CSFV-E2 proteins, in contrast cells infected with the modified viruses vR26_TAV and vR26_E2gif, rescued from the recombined BACs, were not detected by these mAbs. Furthermore, as expected, cells infected with the vR26_E2gif were recognized by the anti-BDV mAb (WB166) whereas no staining was observed with this antibody in vR26 infected cells or in cells with vR26_TAV. The mAb WH303 recognizes the CSFV TAV-epitope [18] and the difference in 4 aa between the TAV-epitope and the corresponding sequence from BDV strain \"Gifhorn\" is enough to completely abolish the recognition by this mAb.", "The mAb WH303 recognizes the CSFV TAV-epitope [18] and the difference in 4 aa between the TAV-epitope and the corresponding sequence from BDV strain \"Gifhorn\" is enough to completely abolish the recognition by this mAb. The lack of staining of vR26_TAV infected cells by the WH211 indicated that the TAV-sequence is also important for the epitope recognized by this mAb. Thus, the chimeric pestiviruses, vR26_TAV and vR26_E2gif, containing heterologous E2 sequences can be readily discriminated from the vR26 using specific anti-E2 monoclonal antibodies.", "Thus, the chimeric pestiviruses, vR26_TAV and vR26_E2gif, containing heterologous E2 sequences can be readily discriminated from the vR26 using specific anti-E2 monoclonal antibodies. These new chimeric pestiviruses represents Cstrain based marked vaccine candidates with the characteristics desired for safe and efficacious DIVA vaccines against CSFV. Indeed, vR26_E2gif vaccinated pigs could be efficiently discriminated from C-strain vaccinated pigs and from CSFV infected pigs using CSFV-E2 specific antibody ELISAs (Rasmussen et al., unpublished results).", "Indeed, vR26_E2gif vaccinated pigs could be efficiently discriminated from C-strain vaccinated pigs and from CSFV infected pigs using CSFV-E2 specific antibody ELISAs (Rasmussen et al., unpublished results). Nucleotide sequence data for the pBeloR26 showed a number of changes from the published reference sequence for \"C-strain Riems\". Some of these differences are present in the cDNA derived from the vaccine stock at a detectable level whereas others may represent low-level variants within the cDNA or errors introduced by the RT-PCR amplification.", "Some of these differences are present in the cDNA derived from the vaccine stock at a detectable level whereas others may represent low-level variants within the cDNA or errors introduced by the RT-PCR amplification. Full-length sequencing revealed that no changes occurred in the cDNA during extensive propagation in E. coli DH10B of the pBeloR26 and the E2chimeric derivative, pBeloR26_E2gif, indicating a very high stability of these BAC-cloned CSFV cDNAs. This is essential if this system is to be useful for cloning and sequence manipulation, and contrasts with stability problems encountered with conventional plasmids containing fulllength pestivirus cDNAs [31] .", "This is essential if this system is to be useful for cloning and sequence manipulation, and contrasts with stability problems encountered with conventional plasmids containing fulllength pestivirus cDNAs [31] . The stability of these BACs is consistent with previous reports on the stability of BACs containing other viruses of the family Flaviviridae in E. coli [8, 10] . Extensive passaging of the rescued vR26 and the chimeric virus derivative, vR26_E2gif, resulted in a change at nucleotide position A10665G (resulting in the predicted aa" ]

[ "BACKGROUND: Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria.", "This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. RESULTS: A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria.", "Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that modifications in the E2 protein coding sequence were stably maintained. A single amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence.", "A single amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence. CONCLUSIONS: These results show that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses. Text: Bacterial artificial chromosomes (BACs) are ideally suited for the stable maintenance of large DNA sequences derived from viral genomes [1] .", "Text: Bacterial artificial chromosomes (BACs) are ideally suited for the stable maintenance of large DNA sequences derived from viral genomes [1] . A considerable number of BAC systems have been established for large DNA viruses; in particular many different herpesvirus genomes have been cloned into BACs (for review see [2] ). The first BAC systems using RNA virus cDNAs were described for coronaviruses [3] [4] [5] [6] and recently the first BAC containing a full-length cDNA for a negative-stranded RNA virus was described [7] .", "The first BAC systems using RNA virus cDNAs were described for coronaviruses [3] [4] [5] [6] and recently the first BAC containing a full-length cDNA for a negative-stranded RNA virus was described [7] . Similarly, cDNAs corresponding to the full-length genomes of members of the Flaviviridae family (Japanese encephalitis virus [8] and Dengue virus [9] ) have been inserted into BACs. BACs containing full-length cDNAs of pestiviruses (also within the Flaviviridae), including bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) have recently been established [10, 11] .", "BACs containing full-length cDNAs of pestiviruses (also within the Flaviviridae), including bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) have recently been established [10, 11] . Infectious pestiviruses can be rescued using RNA transcripts derived from these BACs. The pestiviruses have single stranded positive sense RNA genomes, about 12.3 kb in length, which includes a single long open reading frame, encoding a large polyprotein, flanked by 5′ and 3′ untranslated regions (UTRs) that are critical for autonomous replication of the genome [12, 13] .", "The pestiviruses have single stranded positive sense RNA genomes, about 12.3 kb in length, which includes a single long open reading frame, encoding a large polyprotein, flanked by 5′ and 3′ untranslated regions (UTRs) that are critical for autonomous replication of the genome [12, 13] . The polyprotein is cleaved by cellular and viral proteases into four structural proteins (nucleocapsid protein C, envelope glycoproteins E rns , E1 and E2) and eight nonstructural proteins (N pro , p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). The availability of genetically defined and stable pestivirus BACs facilitates the functional study of viral proteins or RNA structures and also the development of new marker vaccine candidates.", "The availability of genetically defined and stable pestivirus BACs facilitates the functional study of viral proteins or RNA structures and also the development of new marker vaccine candidates. Several CSFV vaccines with marker properties based on chimeric pestiviruses have been developed over the years [14] . In particular, chimeric pestiviruses with substitution of the entire E2 protein have been described [15] [16] [17] but also mutants with more subtle modifications, such as the modification of the important TAV-epitope [18] within the CSFV-E2 protein [19, 20] are promising marker vaccine candidates.", "In particular, chimeric pestiviruses with substitution of the entire E2 protein have been described [15] [16] [17] but also mutants with more subtle modifications, such as the modification of the important TAV-epitope [18] within the CSFV-E2 protein [19, 20] are promising marker vaccine candidates. Manipulation of BACs using traditional cloning procedures can be difficult (e.g. because of a lack of convenient restriction enzyme sites) and thus a range of methodologies that apply bacterial genetics, including homologous recombination (e.g.", "because of a lack of convenient restriction enzyme sites) and thus a range of methodologies that apply bacterial genetics, including homologous recombination (e.g. Red/ET homologous recombineering) within the E. coli host, have been developed (for review, see [21] ). The use of homologous recombination allows site-directed mutagenesis of BACs [22] and, by employing a counterselection scheme, specific modifications can be obtained without leaving residual \"foreign\" sequences [23] .", "The use of homologous recombination allows site-directed mutagenesis of BACs [22] and, by employing a counterselection scheme, specific modifications can be obtained without leaving residual \"foreign\" sequences [23] . The main advantage of this method is that there are no target limitations (e.g. based on size or location) and no need for suitable restriction sites.", "based on size or location) and no need for suitable restriction sites. The integration of the modified sequence is performed in vivo (within E. coli) thereby potentially being more accurate than in vitro approaches like PCR-based methods. Although in vitro cloning approaches based on the use of high-fidelity polymerases for PCR amplification have significantly improved in recent years, the use of in vivo approaches should allow a more accurate method of mutagenesis due to the use of the cells own high-fidelity replication system which includes proof reading.", "Although in vitro cloning approaches based on the use of high-fidelity polymerases for PCR amplification have significantly improved in recent years, the use of in vivo approaches should allow a more accurate method of mutagenesis due to the use of the cells own high-fidelity replication system which includes proof reading. Whereas BAC recombination has been commonly used for modifying DNA viruses, there are only very few reports about the use of this technology for RNA viruses [7, 24, 25] . Here, a generally applicable strategy for the manipulation and rescue of chimeric pestiviruses from BACs is described as a model, and the flexibility of this approach is demonstrated by generating different modifications in the viral cDNA of the new CSFV-BAC, pBeloR26, derived from the modified live vaccine strain \"C-strain Riems\".", "Here, a generally applicable strategy for the manipulation and rescue of chimeric pestiviruses from BACs is described as a model, and the flexibility of this approach is demonstrated by generating different modifications in the viral cDNA of the new CSFV-BAC, pBeloR26, derived from the modified live vaccine strain \"C-strain Riems\". The targeted recombination-mediated mutagenesis described here includes the substitution of the 9 amino acid (aa) linear TAV-epitope (TAVSPTTLR) present in the E2 protein with the corresponding region (TTVSTSTLA) of a heterologous pestivirus (border disease virus, BDV, strain \"Gifhorn\") and also the replacement of the entire CSFV E2 protein coding region with the whole E2 coding region from the same BDV, to generate marked vaccine viruses that can be discriminated using specific anti-E2 monoclonal antibodies. The genetic stabilities of both the BAC constructs (within E. coli) and the rescued viruses have also been assessed.", "The genetic stabilities of both the BAC constructs (within E. coli) and the rescued viruses have also been assessed. Porcine kidney (PK15) and sheep fetal thymoid (SFT-R) cells were grown at 37°C (with 5% (v/v) CO 2 ) in Dulbecco's minimal essential medium (DMEM) supplemented with 5% (v/v) pestivirus-free fetal calf serum. Virus from a bait containing the modified live vaccine CSFV \"C-strain Riems\" (Riemser Arzneimittel AG, Germany) was propagated once in PK15 cells and termed vRiemser.", "Virus from a bait containing the modified live vaccine CSFV \"C-strain Riems\" (Riemser Arzneimittel AG, Germany) was propagated once in PK15 cells and termed vRiemser. RNA obtained from BDV strain \"Gifhorn\" [26] was used for amplification of the Gifhorn E2-coding sequence. Oligonucleotide primers used are listed in Additional file 1: Table S1 .", "Oligonucleotide primers used are listed in Additional file 1: Table S1 . The BAC construct, pBeloR26, was constructed using the long RT-PCR method as previously described [11] using RNA derived from the \"C-strain Riems\". Briefly, full-length viral cDNAs flanked by NotI sites were amplified by long RT-PCR using primers 5′Cstrain_T7_Not1 (which includes a T7 promotor for in vitro transcription, a NotI site and a region corresponding to the first 44 nt of the genome) and 3′CSFV_Not1 (that contains a NotI site and sequence complementary to the 3′-terminal 35 nt of the genome that are conserved among many CSFVs including the Cstrain).", "Briefly, full-length viral cDNAs flanked by NotI sites were amplified by long RT-PCR using primers 5′Cstrain_T7_Not1 (which includes a T7 promotor for in vitro transcription, a NotI site and a region corresponding to the first 44 nt of the genome) and 3′CSFV_Not1 (that contains a NotI site and sequence complementary to the 3′-terminal 35 nt of the genome that are conserved among many CSFVs including the Cstrain). The product (ca. 12.3 kbp) was digested with NotI and inserted into similarly digested pBeloBAC11 (New England Biolabs, GenBank accession U51113).", "12.3 kbp) was digested with NotI and inserted into similarly digested pBeloBAC11 (New England Biolabs, GenBank accession U51113). All BACs were modified and maintained in E. coli DH10B cells (Invitrogen) grown at 37°C in LB medium containing chloramphenicol (Cam, 15 μg/ml). The electroporation of bacteria was performed in 0.1 cm cuvettes using 1 pulse at 1800 V, 25 μF and 200 Ω in a Gene Pulser Xcell (Bio-Rad).", "The electroporation of bacteria was performed in 0.1 cm cuvettes using 1 pulse at 1800 V, 25 μF and 200 Ω in a Gene Pulser Xcell (Bio-Rad). BACs to be used as templates for long PCR or for screening by restriction enzyme digestion were purified from 4 ml overnight cultures of E. coli DH10B using the ZR BAC DNA Miniprep Kit (Zymo Research). BACs required for direct genome sequencing were purified from 500 ml cultures using the Large-construct kit (Qiagen).", "BACs required for direct genome sequencing were purified from 500 ml cultures using the Large-construct kit (Qiagen). Modifications to the full-length CSFV cDNA were accomplished in E. coli DH10B (streptomycin resistant, Strep R ) using the Counter Selection BAC Modification Kit (Gene Bridges, Heidelberg, Germany). The Red/ET recombination involved three steps (i-iii).", "The Red/ET recombination involved three steps (i-iii). Step i) the temperature-sensitive pRedET expression plasmid (Gene Bridges) was introduced into electroporationcompetent E.coli DH10B cells containing the parental BAC (phenotype Cam R , Strep R ). The pRedET expresses the phage lambda proteins redα, redβ and redγ, under control of the arabinose-inducible pBAD promoter, allowing homologous recombination to occur.", "The pRedET expresses the phage lambda proteins redα, redβ and redγ, under control of the arabinose-inducible pBAD promoter, allowing homologous recombination to occur. Immediately after electroporation, pre-warmed LB medium without antibiotics (1 ml) was added to the cells which were then incubated at 30°C for 1 hour, prior to spreading onto agar plates containing Cam (15 μg/ml) and tetracycline (Tet) (3 μg/ml) and then incubated at 30°C overnight to maintain the pRedET. The presence of the pRedET plasmid (conferring Tet R ) was verified by visual inspection of BAC-DNA preparations from the Cam R /Tet R colonies using agarose gel electrophoresis.", "The presence of the pRedET plasmid (conferring Tet R ) was verified by visual inspection of BAC-DNA preparations from the Cam R /Tet R colonies using agarose gel electrophoresis. Step ii) counter-selection marker cassettes with an extra NotI site for screening purposes (rpsL-neo, 1325 bp) were amplified by PCR using primers with 30 nt or 50 nt extensions that were homologous to the target site in the BAC using the rpsL-neo plasmid (Gene Bridges) as template and the Phusion hot start II HF DNA polymerase (Thermo Scientific) with cycling conditions as follows: 98°C for 30s, followed by 35 cycles of 98°C for 10s, 60°C for 20s, 72°C for 60s, and 1 cycle at 72°C for 4 min. The PCR products (ca.", "The PCR products (ca. 1400 bp) were isolated on 1% (w/v) TBE agarose gels and purified using a GeneJET gel extraction kit (Thermo Scientific). Samples (30 μl), from an E. coli culture containing pRedET and the parental BAC grown overnight at 30°C in LB media (Cam, Tet), were used to inoculate 1.4 ml of fresh LB media with the same antibiotics to obtain exponentially growing bacteria at 30°C.", "Samples (30 μl), from an E. coli culture containing pRedET and the parental BAC grown overnight at 30°C in LB media (Cam, Tet), were used to inoculate 1.4 ml of fresh LB media with the same antibiotics to obtain exponentially growing bacteria at 30°C. Red/ET recombination proteins were induced by adding 50 μl of 10% (w/v) L-arabinose (Sigma). The PCR product (200 ng) containing the rpsL-neo cassette was introduced into these bacteria using electroporation (as above).", "The PCR product (200 ng) containing the rpsL-neo cassette was introduced into these bacteria using electroporation (as above). Following electroporation, the cells were grown at 37°C for 70 min (to allow recombination) and then selected on plates containing Cam (15 μg/ml), Tet (3 μg/ml) and kanamycin (Kan, 15 μg/ml) overnight at 30°C to maintain the pRedET. Note, the rpsL cassette confers Streptomycin sensitivity (Strep S ) onto the resistant DH10B strain and the neo confers Kanamycin resistance (Kan R ).", "Note, the rpsL cassette confers Streptomycin sensitivity (Strep S ) onto the resistant DH10B strain and the neo confers Kanamycin resistance (Kan R ). The correct phenotype (Cam R , Kan R , Tet R , Strep S ) of the resulting colonies was confirmed by streaking the colonies onto plates containing Cam (15 μg/ml), Tet (3 μg/ml) and Kan (15 μg/ml) and grown at 30°C. Importantly, for the third step, the replacement of the rpsL-neo cassette (using counter-selection), the selected colonies were also streaked onto plates containing Cam (15 μg/ml) plus Strep (50 μg/ml) and shown to be Strep S indicating incorporation of a functional rpsL gene.", "Importantly, for the third step, the replacement of the rpsL-neo cassette (using counter-selection), the selected colonies were also streaked onto plates containing Cam (15 μg/ml) plus Strep (50 μg/ml) and shown to be Strep S indicating incorporation of a functional rpsL gene. The structures of the intermediate BACs were verified by restriction enzyme analysis and sequencing around the inserts. Step iii) the replacement of the rpsL-neo selection cassettes from the intermediate constructs using linear DNA fragments was achieved through counter-selection and Red/ET recombination.", "Step iii) the replacement of the rpsL-neo selection cassettes from the intermediate constructs using linear DNA fragments was achieved through counter-selection and Red/ET recombination. Again, the homologous sequences at the ends of the DNA fragment were used for Red/ET mediated recombination events to replace the rpsL-neo cassette with the sequence of interest. Counterselection against the rpsL-neo cassette (phenotype Cam R , Kan R , Tet R , Strep S ) was employed using media containing Cam (15 μg/ml) and Strep (50 μg/ml) to isolate the required derivatives (phenotype Cam R and Strep R ).", "Counterselection against the rpsL-neo cassette (phenotype Cam R , Kan R , Tet R , Strep S ) was employed using media containing Cam (15 μg/ml) and Strep (50 μg/ml) to isolate the required derivatives (phenotype Cam R and Strep R ). Initially, the intermediate construct, pBeloR26_E2rpsLneo ( Figure 1 ), was generated using Red/ET recombination by insertion of the rpsL-neo cassette with an extra NotI site for screening purposes which was amplified using primers Criems-TAVfor and Criems-TAVrev (Additional file 1: Table S1 ) in place of the TAVSPTTLR coding sequence (27 nt) . Secondly, the rpsL-neo cassette in this intermediate construct was then replaced using counter-selection Red/ ET recombination using a single-stranded oligonucleotide, Riems_TAV_Gifhorn (Additional file 1: Table S1 ) with the same homology arms as used for the rpsL-neo cassette, to introduce the coding sequence for the BDV \"Gifhorn\" epitope sequence (TTVSTSTLA).", "Secondly, the rpsL-neo cassette in this intermediate construct was then replaced using counter-selection Red/ ET recombination using a single-stranded oligonucleotide, Riems_TAV_Gifhorn (Additional file 1: Table S1 ) with the same homology arms as used for the rpsL-neo cassette, to introduce the coding sequence for the BDV \"Gifhorn\" epitope sequence (TTVSTSTLA). The resulting construct was named pBeloR26_TAV (Figure 1 ). The initial intermediate construct (with rpsL-neo) was then used to produce the pBeloR26_E2gif construct ( Figure 1 ).", "The initial intermediate construct (with rpsL-neo) was then used to produce the pBeloR26_E2gif construct ( Figure 1 ). For this, the E2 coding sequence was amplified from cDNA prepared from BDV \"Gifhorn\" RNA using two different primer pairs, one set with 50 nt homology arms (Criems_E2_gifFlong/Criems_ E2_gifRlong) and another with 30 nt homologous sequences (Criems_E2_gifF/Criems_E2_gifR). For generation of BACs with substitution of the entire E2 coding sequences, PCR products consisting of the sequence of interest flanked with homology arms identical to the target area were generated by PCR (as for the rpsLneo cassette).", "For generation of BACs with substitution of the entire E2 coding sequences, PCR products consisting of the sequence of interest flanked with homology arms identical to the target area were generated by PCR (as for the rpsLneo cassette). For making constructs with substitution of shorter sequences (e.g. the TAV-epitope), the recombination was achieved using synthetic single stranded oligonucleotides rather than PCR products.", "the TAV-epitope), the recombination was achieved using synthetic single stranded oligonucleotides rather than PCR products. Pre-heating of single stranded oligonucleotides at 95°C for 2 min followed by snap-freezing, prior to electroporation, empirically showed the best results. In each case, the DNA molecules were introduced into E. coli containing the BAC derivatives including the rpsL-neo cassettes together with the pRedET plasmid by electroporation as described above.", "In each case, the DNA molecules were introduced into E. coli containing the BAC derivatives including the rpsL-neo cassettes together with the pRedET plasmid by electroporation as described above. The structures of the modified BACs were verified by restriction enzyme analysis and subsequent full-genome sequencing (see below). BAC DNA (1 μg) was linearized with NotI or 1 μl BAC DNA was used as template for long PCR amplification using primers 5′C-strain_T7_Not1 and 3′CSFV (Additional file 1: Table S1 ).", "BAC DNA (1 μg) was linearized with NotI or 1 μl BAC DNA was used as template for long PCR amplification using primers 5′C-strain_T7_Not1 and 3′CSFV (Additional file 1: Table S1 ). Linearized BACs or PCR products were purified with the GeneJet PCR purification kit (Thermo Scientific) and transcribed in vitro using a Megascript T7 kit (Invitrogen). Viruses were rescued from RNA transcripts (1 to 5 μg) by electroporation of porcine (PK15) or ovine (SFT-R) cells essentially as described previously [24] .", "Viruses were rescued from RNA transcripts (1 to 5 μg) by electroporation of porcine (PK15) or ovine (SFT-R) cells essentially as described previously [24] . Cells were analysed using immunofluorescence microscopy (typically after 3 days) for the expression of NS3 and E2 proteins using specific monoclonal antibodies (mAbs), these were anti-NS3 (WB103/105, pan-pestivirus), anti-CSFV E2 (WH211, WH303, both CSFV specific) and anti-BDV E2 (WB166, BVDV/BDV specific) (AHVLA Scientific, United Kingdom) together with Alexa 488 conjugated goat antimouse IgG antibody (Molecular Probes, Invitrogen). The nuclei of cells were visualized using DAPI (Vector Laboratories) and images were recorded using a BX63 fluorescence microscope (Olympus).", "The nuclei of cells were visualized using DAPI (Vector Laboratories) and images were recorded using a BX63 fluorescence microscope (Olympus). For peroxidase staining, cells were fixed and stained for the presence of pestivirus antigens using biotinylated pig anti-CSFV/BVDV polyclonal IgG followed by avidin-conjugated horseradish peroxidase (eBioscience) as previously described [27] . The same staining procedure was also performed using the anti-E2 mAbs.", "The same staining procedure was also performed using the anti-E2 mAbs. Samples containing virus-positive cells were passaged onto new cells. Virus growth curves were generated as previously described [24] . Briefly, PK15 or SFT-R cells were infected at a multiplicity of infection (MOI) of 0.1 pfu/cell and grown for three days.", "Briefly, PK15 or SFT-R cells were infected at a multiplicity of infection (MOI) of 0.1 pfu/cell and grown for three days. BAC DNAs (5 μg), purified using the Large-construct kit (Qiagen), or PCR products (1 μg) amplified from viral cDNA or from BACs using the long PCR method (as above) were consensus sequenced using a 454 FLX (Roche) or an Ion PGM (Life Technologies). Both Newbler (Roche) and the bwa.bwasw alignment algorithm [28] were used for mapping the reads to the expected sequence.", "Both Newbler (Roche) and the bwa.bwasw alignment algorithm [28] were used for mapping the reads to the expected sequence. A combination of Samtools [29] and LoFreq SNV-caller [30] was used for downstream single nucleotide variant (SNV) analysis. Finally, clone consensus sequences were aligned using MAFFT in the Geneious software platform (Biomatters).", "Finally, clone consensus sequences were aligned using MAFFT in the Geneious software platform (Biomatters). Generation of a BAC containing full-length cDNA corresponding to the modified live vaccine \"C-strain Riems\"\n\nBACs containing the full-length cDNA corresponding to the parental vRiemser (\"C-strain Riems\") were constructed according to the method described previously for the \"Paderborn\" strain of CSFV [11] . BACs containing the complete CSFV cDNAs were identified by restriction Figure 1 Schematic representation of the CSFV genome organization and the BACs constructed and used in this study.", "BACs containing the complete CSFV cDNAs were identified by restriction Figure 1 Schematic representation of the CSFV genome organization and the BACs constructed and used in this study. Nucleotide (nt) and amino acid (aa) positions within R26 for the 5′ and 3′ termini together with the translational start and stop codons of the polyprotein coding region plus cleavage sites used to make the individual proteins (N pro , C, E rns , E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) are indicated. Insertion of the rpsL-neo in place of the TAV-epitope within CSFV E2 for the intermediate construct (R26_rpsLneo) and the subsequent replacement with the TTVSTSTLA sequence (R26_TAV) and the complete substitution of the E2 sequence (R26_E2gif) are shown.", "Insertion of the rpsL-neo in place of the TAV-epitope within CSFV E2 for the intermediate construct (R26_rpsLneo) and the subsequent replacement with the TTVSTSTLA sequence (R26_TAV) and the complete substitution of the E2 sequence (R26_E2gif) are shown. Names of BAC constructs begin with \"pBelo\" and rescued viruses with \"v\" (e.g. pBeloR26 and vR26).", "pBeloR26 and vR26). Cell culture passage no. of virus is indicated with \"/P\" (e.g. vR26/P-4). digest analysis and following linearization by NotI, RNA transcripts were produced and electroporated into PK15 cells.", "digest analysis and following linearization by NotI, RNA transcripts were produced and electroporated into PK15 cells. This screening resulted in the identification of a BAC containing a cDNA insert of 12316 nt, pBeloR26 (Figure 1) , which yielded infectious virus, termed vR26, that could be propagated in SFT-R cells (Figure 2 , upper panels) and in PK15 cells (Figure 3 ). The rescued vR26 displayed higher growth rate at the early stage (about 10fold difference in virus yield at 24 h) compared to the parental vaccine virus, but after 48 hours similar virus titres were obtained (Figure 3 ).", "The rescued vR26 displayed higher growth rate at the early stage (about 10fold difference in virus yield at 24 h) compared to the parental vaccine virus, but after 48 hours similar virus titres were obtained (Figure 3 ). Full-genome sequencing of the cloned BAC template, pBeloR26, revealed a number of differences throughout the genome when compared to the full-length consensus sequence of the cDNA used for the cloning procedure (see Table 1 ). These differences are non-representative variants within the cDNA.", "These differences are non-representative variants within the cDNA. Overall, the BAC sequence differed from the cDNA sequence in 18 positions, 9 of these lead to predicted amino acid substitutions within the polyprotein; one in each of N pro , E rns , E1, E2 and NS3 and four amino acid substitutions in NS5B (Table 1) . When compared to the published reference sequence (GenBank accession AY259122.1), the pBeloR26 BAC sequence differed at an additional 11 positions, 1 of these lead to a predicted amino acid substitution and there was one large insertion (27 nt) in the hypervariable region of the 3′-UTR (Additional file 2: Table S2 ).", "When compared to the published reference sequence (GenBank accession AY259122.1), the pBeloR26 BAC sequence differed at an additional 11 positions, 1 of these lead to a predicted amino acid substitution and there was one large insertion (27 nt) in the hypervariable region of the 3′-UTR (Additional file 2: Table S2 ). To determine the utility of the targeted recombinationmediated mutagenesis system for pestiviruses, two different modifications of the E2 protein coding sequence within pBeloR26 were generated using the Red/ET recombination methodology. Initially, the sequence encoding the linear TAV-epitope (TAVSPTTLR) within the CSFV-E2 was substituted with the sequence encoding the corresponding region (encoding TTVSTSTLA) from the BDV strain \"Gifhorn\" as described in the Materials and Methods section.", "Initially, the sequence encoding the linear TAV-epitope (TAVSPTTLR) within the CSFV-E2 was substituted with the sequence encoding the corresponding region (encoding TTVSTSTLA) from the BDV strain \"Gifhorn\" as described in the Materials and Methods section. More than 90% of the colonies obtained using this procedure contained the required BAC\n\nAnti-CSFV E2 (WH211) Figure 2 Antibody reaction patterns of pestivirus infected cells. SFT-R cells were infected with vR26 and its two derivatives vR26_E2gif and vR26_TAV plus vGifhorn [26] .", "SFT-R cells were infected with vR26 and its two derivatives vR26_E2gif and vR26_TAV plus vGifhorn [26] . After 72 h, the cells were fixed and stained with monoclonal antibodies against the NS3 protein (WB103/105, left column), the CSFV E2 protein (WH303 and WH211, middle columns) and the BDV E2 protein (WB166, right column) as indicated and viewed using a fluorescence microscope. structure as determined by NotI digestions.", "structure as determined by NotI digestions. The complete genome sequences of the CSFV cDNA within two selected BACs, designated pBeloR26_TAV have been verified (data not shown). In addition, the complete coding sequence (1119 nt) for the CSFV-E2 protein was substituted by the corresponding sequence from BDV \"Gifhorn\".", "In addition, the complete coding sequence (1119 nt) for the CSFV-E2 protein was substituted by the corresponding sequence from BDV \"Gifhorn\". Again more than 90% of the colonies obtained contained the required BAC and the same proportion of correctly recombined BACs was obtained using either 30 nt or 50 nt homology arms. The chimeric BAC was designated, pBeloR26_E2gif and the complete virus genome sequence (cDNA) was verified (data not shown).", "The chimeric BAC was designated, pBeloR26_E2gif and the complete virus genome sequence (cDNA) was verified (data not shown). After electroporation with RNA transcripts derived from either pBeloR26_TAV or pBeloR26_E2gif a large number of CSFV NS3-positive cells could be observed (data not shown) and chimeric virus stocks, termed vR26_TAV and vR26_E2gif, were generated after further passages in cells. Cells infected with these viruses and with the parental vR26 and vGifhorn strains were all stained with mAbs directed against the NS3 protein ( Figure 2 ).", "Cells infected with these viruses and with the parental vR26 and vGifhorn strains were all stained with mAbs directed against the NS3 protein ( Figure 2 ). However, in contrast to the parental vR26 virus, the chimeric viruses rescued from the recombined BACs were not recognized by anti-E2 mAbs specific for the CSFV-E2 proteins ( Figure 2 ) and thus, consistent with their structure, displayed the same antibody reaction pattern as vGifhorn. Two different anti-CSFV E2 mAbs, WH211 and WH303, were used for the staining and the latter has been shown previously to target the TAV-epitope [18] .", "Two different anti-CSFV E2 mAbs, WH211 and WH303, were used for the staining and the latter has been shown previously to target the TAV-epitope [18] . As anticipated, cells infected with either the vGifhorn or with the chimeric vR26_E2gif could be shown to express the \"Gifhorn\" E2 protein using staining with an anti-BDV mAb ( Figure 2 ). The presence of the BDV epitope TTVSTSTLA in vR26_ TAV was insufficient to permit efficient recognition by this anti-BDV mab, although a weak signal was observed in some cells.", "The presence of the BDV epitope TTVSTSTLA in vR26_ TAV was insufficient to permit efficient recognition by this anti-BDV mab, although a weak signal was observed in some cells. The BAC constructs pBeloR26 and pBeloR26_E2gif were analysed for the genetic stability of the cDNA to determine the suitability of the BAC vector for maintaining full-length pestivirus cDNAs. E. coli DH10B cells containing the BACs were passaged 15 times, by overnight growth, and the complete viral cDNAs within the BACs were sequenced after the 1st and the 15th passage.", "E. coli DH10B cells containing the BACs were passaged 15 times, by overnight growth, and the complete viral cDNAs within the BACs were sequenced after the 1st and the 15th passage. No mutations were observed within the 12316 nt virus cDNA sequences after this extensive propagation of the BACs in the bacterial host, indicating a highly stable system for the maintenance of complete pestivirus cDNA sequences. The viruses, vR26 and vR26_E2gif, rescued from their respective BAC constructs, were also tested for their genetic stability within mammalian cells.", "The viruses, vR26 and vR26_E2gif, rescued from their respective BAC constructs, were also tested for their genetic stability within mammalian cells. Linearized BAC DNA was transcribed in vitro and the RNA was electroporated into PK15 cells. Three days after electroporation the cells were stained with the anti-NS3 antibody to detect the presence of replicating virus.", "Three days after electroporation the cells were stained with the anti-NS3 antibody to detect the presence of replicating virus. Samples containing virus positive cells were passaged onto new cells, this process \n\n*Nt position 10665 in vR26/P-12 is reverted from A to G as in the parental cDNA. was repeated for 12 separate passages (each of three days).", "was repeated for 12 separate passages (each of three days). The virus titre (as TCID 50 /ml) was determined for each passage. Passage of the rescued vR26_E2gif chimeric virus in PK15 cells resulted in rapidly decreasing virus titres and was discontinued after the 2nd passage ( Figure 4A ).", "Passage of the rescued vR26_E2gif chimeric virus in PK15 cells resulted in rapidly decreasing virus titres and was discontinued after the 2nd passage ( Figure 4A ). Instead, further passage of this chimeric virus was performed in ovine SFT-R cells (the preferred cell type for BDV) and resulted in much higher titers of the chimeric virus. Virus titers reached more than 10 6 TCID 50 /ml after the 1st passage and remained stable for 12 passages ( Figure 4A ).", "Virus titers reached more than 10 6 TCID 50 /ml after the 1st passage and remained stable for 12 passages ( Figure 4A ). The rescued vR26 was also efficiently propagated on the SFT-R cells but maintained a slightly lower titer than the vR26_E2gif chimeric virus ( Figure 4A ). To check that the viruses retained their antibody reaction properties ( Figure 2 ) after these passages, cells were infected with viruses from the 12th SFT-R cell culture passage (termed vR26/P-12 and vR26_E2gif/P-12) and stained with a polyclonal anti-pestivirus serum and with specific mAbs directed against the CSFV-E2 and BDV-E2 proteins ( Figure 4B ).", "To check that the viruses retained their antibody reaction properties ( Figure 2 ) after these passages, cells were infected with viruses from the 12th SFT-R cell culture passage (termed vR26/P-12 and vR26_E2gif/P-12) and stained with a polyclonal anti-pestivirus serum and with specific mAbs directed against the CSFV-E2 and BDV-E2 proteins ( Figure 4B ). Cells infected with either the vR26/P-12 or the chimeric vR26_E2gif/P-12 were each detected by the polyclonal anti-pestivirus serum as expected. The anti-CSFV-E2 mAb specifically detected cells infected with vR26/P-12 but not cells infected by the chimeric virus containing the BDV-E2 protein (consistent with the results shown in Figure 2 ).", "The anti-CSFV-E2 mAb specifically detected cells infected with vR26/P-12 but not cells infected by the chimeric virus containing the BDV-E2 protein (consistent with the results shown in Figure 2 ). In contrast, the anti-BDV-E2 mAb specifically detected infection by the vR26_E2gif/P-12 and did not recognize cells infected with vR26/P-12. Each result is in accord with the structure of the viruses.", "Each result is in accord with the structure of the viruses. The 4th passage of vR26 (vR26/P-4) displayed a slower growth rate than the virus obtained after 12 passages (see Figure 5A ). It also had a reduced growth rate compared to both the vR26_E2gif/P-4 and vR26_E2gif/P-12.", "It also had a reduced growth rate compared to both the vR26_E2gif/P-4 and vR26_E2gif/P-12. The fulllength sequence of pBeloR26 had revealed ten non-silent mutations compared to the reference sequence (AY25 9122.1) for this virus (Additional file 2: Table S2 ). Any of these mutations could be responsible for the impaired growth acting alone or in concert.", "Any of these mutations could be responsible for the impaired growth acting alone or in concert. For further investigation of this issue, full length cDNAs prepared from vR26/ P-4, vR26/P-12, vR26_E2gif/P-4 and vR26_E2gif/P-12 were deep-sequenced using both the 454 FLX and Ion PGM platforms for comparison and to determine the quasispecies distribution (Additional file 3: Figure S1 and Additional file 4: Figure S2 ). Sequencing data from both platforms revealed that both the vR26/P-12 and vR26_E2gif/P-12 were close to 100% changed at nt position A10665G compared to the BAC clones (resulting in the predicted amino acid substitution D3431G within the NS5B protein, the RNAdependent RNA polymerase, see Figure 5B ).", "Sequencing data from both platforms revealed that both the vR26/P-12 and vR26_E2gif/P-12 were close to 100% changed at nt position A10665G compared to the BAC clones (resulting in the predicted amino acid substitution D3431G within the NS5B protein, the RNAdependent RNA polymerase, see Figure 5B ). This adaptation is a reversion back to the consensus cDNA sequence of the parental vaccine virus, vRiemser (Additional file 2: Table S2 ). Additionally, vR26/P-4 and vR26_E2gif/P-4 already showed evidence for this reversion being present within the population.", "Additionally, vR26/P-4 and vR26_E2gif/P-4 already showed evidence for this reversion being present within the population. For vR26/P-4, the level of reversion was 57%, while for vR26_E2gif/P-4 the extent of change was 73% (see Figure 5B ). In this study, we have established the first BAC containing the full-length cDNA of a CSFV vaccine strain.", "In this study, we have established the first BAC containing the full-length cDNA of a CSFV vaccine strain. The BAC differed from the parental cDNA sequence in 18 positions leading to 9 aa substitutions ( Table 1 ). The method that has been used for the generation of pBeloR26 is based on full genome amplification of cDNA followed by direct cloning to obtain the BACs [11] .", "The method that has been used for the generation of pBeloR26 is based on full genome amplification of cDNA followed by direct cloning to obtain the BACs [11] . This approach results in cDNA clones that reflect the quasispecies composition of the parental viral RNA and thus it is not guaranteed to obtain cDNA clones corresponding to the consensus sequence of the cDNA used. However, it is possible to correct the mutations using the BAC recombination approach if a consensus clone is needed.", "However, it is possible to correct the mutations using the BAC recombination approach if a consensus clone is needed. To demonstrate the utility of the Red/ET mediated recombination method we have generated a series of modified BACs derived from this CSFV full-length cDNA. These include BACs with substitution of the linear TAV-epitope present in the E2 protein and also BACs with substitution of the complete E2 protein with heterologous pestivirus sequences.", "These include BACs with substitution of the linear TAV-epitope present in the E2 protein and also BACs with substitution of the complete E2 protein with heterologous pestivirus sequences. We have also used the same approach for a range of different targeted modifications within CSFV BACs including specific deletions and substitutions in the 5′UTR of CSFV [24] and for insertions of heterologous reporter sequences into CSFV replicons [25] . Using Red/ET recombinationmediated mutagenesis for the targeted design, the work can be expedited and focused, in principal, on any sequence within the viral genome and is not dependent on the use of internal restriction sites.", "Using Red/ET recombinationmediated mutagenesis for the targeted design, the work can be expedited and focused, in principal, on any sequence within the viral genome and is not dependent on the use of internal restriction sites. The results demonstrate that Red/ ET recombination-mediated mutagenesis of pestivirus BAC cDNAs provides a useful tool for advancing the construction of modified pestiviruses. Cells infected with the parental vR26 virus were recognized by the two anti-E2 mAbs (WH211 and WH303) specific for the CSFV-E2 proteins, in contrast cells infected with the modified viruses vR26_TAV and vR26_E2gif, rescued from the recombined BACs, were not detected by these mAbs.", "Cells infected with the parental vR26 virus were recognized by the two anti-E2 mAbs (WH211 and WH303) specific for the CSFV-E2 proteins, in contrast cells infected with the modified viruses vR26_TAV and vR26_E2gif, rescued from the recombined BACs, were not detected by these mAbs. Furthermore, as expected, cells infected with the vR26_E2gif were recognized by the anti-BDV mAb (WB166) whereas no staining was observed with this antibody in vR26 infected cells or in cells with vR26_TAV. The mAb WH303 recognizes the CSFV TAV-epitope [18] and the difference in 4 aa between the TAV-epitope and the corresponding sequence from BDV strain \"Gifhorn\" is enough to completely abolish the recognition by this mAb.", "The mAb WH303 recognizes the CSFV TAV-epitope [18] and the difference in 4 aa between the TAV-epitope and the corresponding sequence from BDV strain \"Gifhorn\" is enough to completely abolish the recognition by this mAb. The lack of staining of vR26_TAV infected cells by the WH211 indicated that the TAV-sequence is also important for the epitope recognized by this mAb. Thus, the chimeric pestiviruses, vR26_TAV and vR26_E2gif, containing heterologous E2 sequences can be readily discriminated from the vR26 using specific anti-E2 monoclonal antibodies.", "Thus, the chimeric pestiviruses, vR26_TAV and vR26_E2gif, containing heterologous E2 sequences can be readily discriminated from the vR26 using specific anti-E2 monoclonal antibodies. These new chimeric pestiviruses represents Cstrain based marked vaccine candidates with the characteristics desired for safe and efficacious DIVA vaccines against CSFV. Indeed, vR26_E2gif vaccinated pigs could be efficiently discriminated from C-strain vaccinated pigs and from CSFV infected pigs using CSFV-E2 specific antibody ELISAs (Rasmussen et al., unpublished results).", "Indeed, vR26_E2gif vaccinated pigs could be efficiently discriminated from C-strain vaccinated pigs and from CSFV infected pigs using CSFV-E2 specific antibody ELISAs (Rasmussen et al., unpublished results). Nucleotide sequence data for the pBeloR26 showed a number of changes from the published reference sequence for \"C-strain Riems\". Some of these differences are present in the cDNA derived from the vaccine stock at a detectable level whereas others may represent low-level variants within the cDNA or errors introduced by the RT-PCR amplification.", "Some of these differences are present in the cDNA derived from the vaccine stock at a detectable level whereas others may represent low-level variants within the cDNA or errors introduced by the RT-PCR amplification. Full-length sequencing revealed that no changes occurred in the cDNA during extensive propagation in E. coli DH10B of the pBeloR26 and the E2chimeric derivative, pBeloR26_E2gif, indicating a very high stability of these BAC-cloned CSFV cDNAs. This is essential if this system is to be useful for cloning and sequence manipulation, and contrasts with stability problems encountered with conventional plasmids containing fulllength pestivirus cDNAs [31] .", "This is essential if this system is to be useful for cloning and sequence manipulation, and contrasts with stability problems encountered with conventional plasmids containing fulllength pestivirus cDNAs [31] . The stability of these BACs is consistent with previous reports on the stability of BACs containing other viruses of the family Flaviviridae in E. coli [8, 10] . Extensive passaging of the rescued vR26 and the chimeric virus derivative, vR26_E2gif, resulted in a change at nucleotide position A10665G (resulting in the predicted aa" ]

[ "BACKGROUND: Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria.", "This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. RESULTS: A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria.", "Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that modifications in the E2 protein coding sequence were stably maintained. A single amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence.", "A single amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence. CONCLUSIONS: These results show that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses. Text: Bacterial artificial chromosomes (BACs) are ideally suited for the stable maintenance of large DNA sequences derived from viral genomes [1] .", "Text: Bacterial artificial chromosomes (BACs) are ideally suited for the stable maintenance of large DNA sequences derived from viral genomes [1] . A considerable number of BAC systems have been established for large DNA viruses; in particular many different herpesvirus genomes have been cloned into BACs (for review see [2] ). The first BAC systems using RNA virus cDNAs were described for coronaviruses [3] [4] [5] [6] and recently the first BAC containing a full-length cDNA for a negative-stranded RNA virus was described [7] .", "The first BAC systems using RNA virus cDNAs were described for coronaviruses [3] [4] [5] [6] and recently the first BAC containing a full-length cDNA for a negative-stranded RNA virus was described [7] . Similarly, cDNAs corresponding to the full-length genomes of members of the Flaviviridae family (Japanese encephalitis virus [8] and Dengue virus [9] ) have been inserted into BACs. BACs containing full-length cDNAs of pestiviruses (also within the Flaviviridae), including bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) have recently been established [10, 11] .", "BACs containing full-length cDNAs of pestiviruses (also within the Flaviviridae), including bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) have recently been established [10, 11] . Infectious pestiviruses can be rescued using RNA transcripts derived from these BACs. The pestiviruses have single stranded positive sense RNA genomes, about 12.3 kb in length, which includes a single long open reading frame, encoding a large polyprotein, flanked by 5′ and 3′ untranslated regions (UTRs) that are critical for autonomous replication of the genome [12, 13] .", "The pestiviruses have single stranded positive sense RNA genomes, about 12.3 kb in length, which includes a single long open reading frame, encoding a large polyprotein, flanked by 5′ and 3′ untranslated regions (UTRs) that are critical for autonomous replication of the genome [12, 13] . The polyprotein is cleaved by cellular and viral proteases into four structural proteins (nucleocapsid protein C, envelope glycoproteins E rns , E1 and E2) and eight nonstructural proteins (N pro , p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). The availability of genetically defined and stable pestivirus BACs facilitates the functional study of viral proteins or RNA structures and also the development of new marker vaccine candidates.", "The availability of genetically defined and stable pestivirus BACs facilitates the functional study of viral proteins or RNA structures and also the development of new marker vaccine candidates. Several CSFV vaccines with marker properties based on chimeric pestiviruses have been developed over the years [14] . In particular, chimeric pestiviruses with substitution of the entire E2 protein have been described [15] [16] [17] but also mutants with more subtle modifications, such as the modification of the important TAV-epitope [18] within the CSFV-E2 protein [19, 20] are promising marker vaccine candidates.", "In particular, chimeric pestiviruses with substitution of the entire E2 protein have been described [15] [16] [17] but also mutants with more subtle modifications, such as the modification of the important TAV-epitope [18] within the CSFV-E2 protein [19, 20] are promising marker vaccine candidates. Manipulation of BACs using traditional cloning procedures can be difficult (e.g. because of a lack of convenient restriction enzyme sites) and thus a range of methodologies that apply bacterial genetics, including homologous recombination (e.g.", "because of a lack of convenient restriction enzyme sites) and thus a range of methodologies that apply bacterial genetics, including homologous recombination (e.g. Red/ET homologous recombineering) within the E. coli host, have been developed (for review, see [21] ). The use of homologous recombination allows site-directed mutagenesis of BACs [22] and, by employing a counterselection scheme, specific modifications can be obtained without leaving residual \"foreign\" sequences [23] .", "The use of homologous recombination allows site-directed mutagenesis of BACs [22] and, by employing a counterselection scheme, specific modifications can be obtained without leaving residual \"foreign\" sequences [23] . The main advantage of this method is that there are no target limitations (e.g. based on size or location) and no need for suitable restriction sites.", "based on size or location) and no need for suitable restriction sites. The integration of the modified sequence is performed in vivo (within E. coli) thereby potentially being more accurate than in vitro approaches like PCR-based methods. Although in vitro cloning approaches based on the use of high-fidelity polymerases for PCR amplification have significantly improved in recent years, the use of in vivo approaches should allow a more accurate method of mutagenesis due to the use of the cells own high-fidelity replication system which includes proof reading.", "Although in vitro cloning approaches based on the use of high-fidelity polymerases for PCR amplification have significantly improved in recent years, the use of in vivo approaches should allow a more accurate method of mutagenesis due to the use of the cells own high-fidelity replication system which includes proof reading. Whereas BAC recombination has been commonly used for modifying DNA viruses, there are only very few reports about the use of this technology for RNA viruses [7, 24, 25] . Here, a generally applicable strategy for the manipulation and rescue of chimeric pestiviruses from BACs is described as a model, and the flexibility of this approach is demonstrated by generating different modifications in the viral cDNA of the new CSFV-BAC, pBeloR26, derived from the modified live vaccine strain \"C-strain Riems\".", "Here, a generally applicable strategy for the manipulation and rescue of chimeric pestiviruses from BACs is described as a model, and the flexibility of this approach is demonstrated by generating different modifications in the viral cDNA of the new CSFV-BAC, pBeloR26, derived from the modified live vaccine strain \"C-strain Riems\". The targeted recombination-mediated mutagenesis described here includes the substitution of the 9 amino acid (aa) linear TAV-epitope (TAVSPTTLR) present in the E2 protein with the corresponding region (TTVSTSTLA) of a heterologous pestivirus (border disease virus, BDV, strain \"Gifhorn\") and also the replacement of the entire CSFV E2 protein coding region with the whole E2 coding region from the same BDV, to generate marked vaccine viruses that can be discriminated using specific anti-E2 monoclonal antibodies. The genetic stabilities of both the BAC constructs (within E. coli) and the rescued viruses have also been assessed.", "The genetic stabilities of both the BAC constructs (within E. coli) and the rescued viruses have also been assessed. Porcine kidney (PK15) and sheep fetal thymoid (SFT-R) cells were grown at 37°C (with 5% (v/v) CO 2 ) in Dulbecco's minimal essential medium (DMEM) supplemented with 5% (v/v) pestivirus-free fetal calf serum. Virus from a bait containing the modified live vaccine CSFV \"C-strain Riems\" (Riemser Arzneimittel AG, Germany) was propagated once in PK15 cells and termed vRiemser.", "Virus from a bait containing the modified live vaccine CSFV \"C-strain Riems\" (Riemser Arzneimittel AG, Germany) was propagated once in PK15 cells and termed vRiemser. RNA obtained from BDV strain \"Gifhorn\" [26] was used for amplification of the Gifhorn E2-coding sequence. Oligonucleotide primers used are listed in Additional file 1: Table S1 .", "Oligonucleotide primers used are listed in Additional file 1: Table S1 . The BAC construct, pBeloR26, was constructed using the long RT-PCR method as previously described [11] using RNA derived from the \"C-strain Riems\". Briefly, full-length viral cDNAs flanked by NotI sites were amplified by long RT-PCR using primers 5′Cstrain_T7_Not1 (which includes a T7 promotor for in vitro transcription, a NotI site and a region corresponding to the first 44 nt of the genome) and 3′CSFV_Not1 (that contains a NotI site and sequence complementary to the 3′-terminal 35 nt of the genome that are conserved among many CSFVs including the Cstrain).", "Briefly, full-length viral cDNAs flanked by NotI sites were amplified by long RT-PCR using primers 5′Cstrain_T7_Not1 (which includes a T7 promotor for in vitro transcription, a NotI site and a region corresponding to the first 44 nt of the genome) and 3′CSFV_Not1 (that contains a NotI site and sequence complementary to the 3′-terminal 35 nt of the genome that are conserved among many CSFVs including the Cstrain). The product (ca. 12.3 kbp) was digested with NotI and inserted into similarly digested pBeloBAC11 (New England Biolabs, GenBank accession U51113).", "12.3 kbp) was digested with NotI and inserted into similarly digested pBeloBAC11 (New England Biolabs, GenBank accession U51113). All BACs were modified and maintained in E. coli DH10B cells (Invitrogen) grown at 37°C in LB medium containing chloramphenicol (Cam, 15 μg/ml). The electroporation of bacteria was performed in 0.1 cm cuvettes using 1 pulse at 1800 V, 25 μF and 200 Ω in a Gene Pulser Xcell (Bio-Rad).", "The electroporation of bacteria was performed in 0.1 cm cuvettes using 1 pulse at 1800 V, 25 μF and 200 Ω in a Gene Pulser Xcell (Bio-Rad). BACs to be used as templates for long PCR or for screening by restriction enzyme digestion were purified from 4 ml overnight cultures of E. coli DH10B using the ZR BAC DNA Miniprep Kit (Zymo Research). BACs required for direct genome sequencing were purified from 500 ml cultures using the Large-construct kit (Qiagen).", "BACs required for direct genome sequencing were purified from 500 ml cultures using the Large-construct kit (Qiagen). Modifications to the full-length CSFV cDNA were accomplished in E. coli DH10B (streptomycin resistant, Strep R ) using the Counter Selection BAC Modification Kit (Gene Bridges, Heidelberg, Germany). The Red/ET recombination involved three steps (i-iii).", "The Red/ET recombination involved three steps (i-iii). Step i) the temperature-sensitive pRedET expression plasmid (Gene Bridges) was introduced into electroporationcompetent E.coli DH10B cells containing the parental BAC (phenotype Cam R , Strep R ). The pRedET expresses the phage lambda proteins redα, redβ and redγ, under control of the arabinose-inducible pBAD promoter, allowing homologous recombination to occur.", "The pRedET expresses the phage lambda proteins redα, redβ and redγ, under control of the arabinose-inducible pBAD promoter, allowing homologous recombination to occur. Immediately after electroporation, pre-warmed LB medium without antibiotics (1 ml) was added to the cells which were then incubated at 30°C for 1 hour, prior to spreading onto agar plates containing Cam (15 μg/ml) and tetracycline (Tet) (3 μg/ml) and then incubated at 30°C overnight to maintain the pRedET. The presence of the pRedET plasmid (conferring Tet R ) was verified by visual inspection of BAC-DNA preparations from the Cam R /Tet R colonies using agarose gel electrophoresis.", "The presence of the pRedET plasmid (conferring Tet R ) was verified by visual inspection of BAC-DNA preparations from the Cam R /Tet R colonies using agarose gel electrophoresis. Step ii) counter-selection marker cassettes with an extra NotI site for screening purposes (rpsL-neo, 1325 bp) were amplified by PCR using primers with 30 nt or 50 nt extensions that were homologous to the target site in the BAC using the rpsL-neo plasmid (Gene Bridges) as template and the Phusion hot start II HF DNA polymerase (Thermo Scientific) with cycling conditions as follows: 98°C for 30s, followed by 35 cycles of 98°C for 10s, 60°C for 20s, 72°C for 60s, and 1 cycle at 72°C for 4 min. The PCR products (ca.", "The PCR products (ca. 1400 bp) were isolated on 1% (w/v) TBE agarose gels and purified using a GeneJET gel extraction kit (Thermo Scientific). Samples (30 μl), from an E. coli culture containing pRedET and the parental BAC grown overnight at 30°C in LB media (Cam, Tet), were used to inoculate 1.4 ml of fresh LB media with the same antibiotics to obtain exponentially growing bacteria at 30°C.", "Samples (30 μl), from an E. coli culture containing pRedET and the parental BAC grown overnight at 30°C in LB media (Cam, Tet), were used to inoculate 1.4 ml of fresh LB media with the same antibiotics to obtain exponentially growing bacteria at 30°C. Red/ET recombination proteins were induced by adding 50 μl of 10% (w/v) L-arabinose (Sigma). The PCR product (200 ng) containing the rpsL-neo cassette was introduced into these bacteria using electroporation (as above).", "The PCR product (200 ng) containing the rpsL-neo cassette was introduced into these bacteria using electroporation (as above). Following electroporation, the cells were grown at 37°C for 70 min (to allow recombination) and then selected on plates containing Cam (15 μg/ml), Tet (3 μg/ml) and kanamycin (Kan, 15 μg/ml) overnight at 30°C to maintain the pRedET. Note, the rpsL cassette confers Streptomycin sensitivity (Strep S ) onto the resistant DH10B strain and the neo confers Kanamycin resistance (Kan R ).", "Note, the rpsL cassette confers Streptomycin sensitivity (Strep S ) onto the resistant DH10B strain and the neo confers Kanamycin resistance (Kan R ). The correct phenotype (Cam R , Kan R , Tet R , Strep S ) of the resulting colonies was confirmed by streaking the colonies onto plates containing Cam (15 μg/ml), Tet (3 μg/ml) and Kan (15 μg/ml) and grown at 30°C. Importantly, for the third step, the replacement of the rpsL-neo cassette (using counter-selection), the selected colonies were also streaked onto plates containing Cam (15 μg/ml) plus Strep (50 μg/ml) and shown to be Strep S indicating incorporation of a functional rpsL gene.", "Importantly, for the third step, the replacement of the rpsL-neo cassette (using counter-selection), the selected colonies were also streaked onto plates containing Cam (15 μg/ml) plus Strep (50 μg/ml) and shown to be Strep S indicating incorporation of a functional rpsL gene. The structures of the intermediate BACs were verified by restriction enzyme analysis and sequencing around the inserts. Step iii) the replacement of the rpsL-neo selection cassettes from the intermediate constructs using linear DNA fragments was achieved through counter-selection and Red/ET recombination.", "Step iii) the replacement of the rpsL-neo selection cassettes from the intermediate constructs using linear DNA fragments was achieved through counter-selection and Red/ET recombination. Again, the homologous sequences at the ends of the DNA fragment were used for Red/ET mediated recombination events to replace the rpsL-neo cassette with the sequence of interest. Counterselection against the rpsL-neo cassette (phenotype Cam R , Kan R , Tet R , Strep S ) was employed using media containing Cam (15 μg/ml) and Strep (50 μg/ml) to isolate the required derivatives (phenotype Cam R and Strep R ).", "Counterselection against the rpsL-neo cassette (phenotype Cam R , Kan R , Tet R , Strep S ) was employed using media containing Cam (15 μg/ml) and Strep (50 μg/ml) to isolate the required derivatives (phenotype Cam R and Strep R ). Initially, the intermediate construct, pBeloR26_E2rpsLneo ( Figure 1 ), was generated using Red/ET recombination by insertion of the rpsL-neo cassette with an extra NotI site for screening purposes which was amplified using primers Criems-TAVfor and Criems-TAVrev (Additional file 1: Table S1 ) in place of the TAVSPTTLR coding sequence (27 nt) . Secondly, the rpsL-neo cassette in this intermediate construct was then replaced using counter-selection Red/ ET recombination using a single-stranded oligonucleotide, Riems_TAV_Gifhorn (Additional file 1: Table S1 ) with the same homology arms as used for the rpsL-neo cassette, to introduce the coding sequence for the BDV \"Gifhorn\" epitope sequence (TTVSTSTLA).", "Secondly, the rpsL-neo cassette in this intermediate construct was then replaced using counter-selection Red/ ET recombination using a single-stranded oligonucleotide, Riems_TAV_Gifhorn (Additional file 1: Table S1 ) with the same homology arms as used for the rpsL-neo cassette, to introduce the coding sequence for the BDV \"Gifhorn\" epitope sequence (TTVSTSTLA). The resulting construct was named pBeloR26_TAV (Figure 1 ). The initial intermediate construct (with rpsL-neo) was then used to produce the pBeloR26_E2gif construct ( Figure 1 ).", "The initial intermediate construct (with rpsL-neo) was then used to produce the pBeloR26_E2gif construct ( Figure 1 ). For this, the E2 coding sequence was amplified from cDNA prepared from BDV \"Gifhorn\" RNA using two different primer pairs, one set with 50 nt homology arms (Criems_E2_gifFlong/Criems_ E2_gifRlong) and another with 30 nt homologous sequences (Criems_E2_gifF/Criems_E2_gifR). For generation of BACs with substitution of the entire E2 coding sequences, PCR products consisting of the sequence of interest flanked with homology arms identical to the target area were generated by PCR (as for the rpsLneo cassette).", "For generation of BACs with substitution of the entire E2 coding sequences, PCR products consisting of the sequence of interest flanked with homology arms identical to the target area were generated by PCR (as for the rpsLneo cassette). For making constructs with substitution of shorter sequences (e.g. the TAV-epitope), the recombination was achieved using synthetic single stranded oligonucleotides rather than PCR products.", "the TAV-epitope), the recombination was achieved using synthetic single stranded oligonucleotides rather than PCR products. Pre-heating of single stranded oligonucleotides at 95°C for 2 min followed by snap-freezing, prior to electroporation, empirically showed the best results. In each case, the DNA molecules were introduced into E. coli containing the BAC derivatives including the rpsL-neo cassettes together with the pRedET plasmid by electroporation as described above.", "In each case, the DNA molecules were introduced into E. coli containing the BAC derivatives including the rpsL-neo cassettes together with the pRedET plasmid by electroporation as described above. The structures of the modified BACs were verified by restriction enzyme analysis and subsequent full-genome sequencing (see below). BAC DNA (1 μg) was linearized with NotI or 1 μl BAC DNA was used as template for long PCR amplification using primers 5′C-strain_T7_Not1 and 3′CSFV (Additional file 1: Table S1 ).", "BAC DNA (1 μg) was linearized with NotI or 1 μl BAC DNA was used as template for long PCR amplification using primers 5′C-strain_T7_Not1 and 3′CSFV (Additional file 1: Table S1 ). Linearized BACs or PCR products were purified with the GeneJet PCR purification kit (Thermo Scientific) and transcribed in vitro using a Megascript T7 kit (Invitrogen). Viruses were rescued from RNA transcripts (1 to 5 μg) by electroporation of porcine (PK15) or ovine (SFT-R) cells essentially as described previously [24] .", "Viruses were rescued from RNA transcripts (1 to 5 μg) by electroporation of porcine (PK15) or ovine (SFT-R) cells essentially as described previously [24] . Cells were analysed using immunofluorescence microscopy (typically after 3 days) for the expression of NS3 and E2 proteins using specific monoclonal antibodies (mAbs), these were anti-NS3 (WB103/105, pan-pestivirus), anti-CSFV E2 (WH211, WH303, both CSFV specific) and anti-BDV E2 (WB166, BVDV/BDV specific) (AHVLA Scientific, United Kingdom) together with Alexa 488 conjugated goat antimouse IgG antibody (Molecular Probes, Invitrogen). The nuclei of cells were visualized using DAPI (Vector Laboratories) and images were recorded using a BX63 fluorescence microscope (Olympus).", "The nuclei of cells were visualized using DAPI (Vector Laboratories) and images were recorded using a BX63 fluorescence microscope (Olympus). For peroxidase staining, cells were fixed and stained for the presence of pestivirus antigens using biotinylated pig anti-CSFV/BVDV polyclonal IgG followed by avidin-conjugated horseradish peroxidase (eBioscience) as previously described [27] . The same staining procedure was also performed using the anti-E2 mAbs.", "The same staining procedure was also performed using the anti-E2 mAbs. Samples containing virus-positive cells were passaged onto new cells. Virus growth curves were generated as previously described [24] . Briefly, PK15 or SFT-R cells were infected at a multiplicity of infection (MOI) of 0.1 pfu/cell and grown for three days.", "Briefly, PK15 or SFT-R cells were infected at a multiplicity of infection (MOI) of 0.1 pfu/cell and grown for three days. BAC DNAs (5 μg), purified using the Large-construct kit (Qiagen), or PCR products (1 μg) amplified from viral cDNA or from BACs using the long PCR method (as above) were consensus sequenced using a 454 FLX (Roche) or an Ion PGM (Life Technologies). Both Newbler (Roche) and the bwa.bwasw alignment algorithm [28] were used for mapping the reads to the expected sequence.", "Both Newbler (Roche) and the bwa.bwasw alignment algorithm [28] were used for mapping the reads to the expected sequence. A combination of Samtools [29] and LoFreq SNV-caller [30] was used for downstream single nucleotide variant (SNV) analysis. Finally, clone consensus sequences were aligned using MAFFT in the Geneious software platform (Biomatters).", "Finally, clone consensus sequences were aligned using MAFFT in the Geneious software platform (Biomatters). Generation of a BAC containing full-length cDNA corresponding to the modified live vaccine \"C-strain Riems\"\n\nBACs containing the full-length cDNA corresponding to the parental vRiemser (\"C-strain Riems\") were constructed according to the method described previously for the \"Paderborn\" strain of CSFV [11] . BACs containing the complete CSFV cDNAs were identified by restriction Figure 1 Schematic representation of the CSFV genome organization and the BACs constructed and used in this study.", "BACs containing the complete CSFV cDNAs were identified by restriction Figure 1 Schematic representation of the CSFV genome organization and the BACs constructed and used in this study. Nucleotide (nt) and amino acid (aa) positions within R26 for the 5′ and 3′ termini together with the translational start and stop codons of the polyprotein coding region plus cleavage sites used to make the individual proteins (N pro , C, E rns , E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) are indicated. Insertion of the rpsL-neo in place of the TAV-epitope within CSFV E2 for the intermediate construct (R26_rpsLneo) and the subsequent replacement with the TTVSTSTLA sequence (R26_TAV) and the complete substitution of the E2 sequence (R26_E2gif) are shown.", "Insertion of the rpsL-neo in place of the TAV-epitope within CSFV E2 for the intermediate construct (R26_rpsLneo) and the subsequent replacement with the TTVSTSTLA sequence (R26_TAV) and the complete substitution of the E2 sequence (R26_E2gif) are shown. Names of BAC constructs begin with \"pBelo\" and rescued viruses with \"v\" (e.g. pBeloR26 and vR26).", "pBeloR26 and vR26). Cell culture passage no. of virus is indicated with \"/P\" (e.g. vR26/P-4). digest analysis and following linearization by NotI, RNA transcripts were produced and electroporated into PK15 cells.", "digest analysis and following linearization by NotI, RNA transcripts were produced and electroporated into PK15 cells. This screening resulted in the identification of a BAC containing a cDNA insert of 12316 nt, pBeloR26 (Figure 1) , which yielded infectious virus, termed vR26, that could be propagated in SFT-R cells (Figure 2 , upper panels) and in PK15 cells (Figure 3 ). The rescued vR26 displayed higher growth rate at the early stage (about 10fold difference in virus yield at 24 h) compared to the parental vaccine virus, but after 48 hours similar virus titres were obtained (Figure 3 ).", "The rescued vR26 displayed higher growth rate at the early stage (about 10fold difference in virus yield at 24 h) compared to the parental vaccine virus, but after 48 hours similar virus titres were obtained (Figure 3 ). Full-genome sequencing of the cloned BAC template, pBeloR26, revealed a number of differences throughout the genome when compared to the full-length consensus sequence of the cDNA used for the cloning procedure (see Table 1 ). These differences are non-representative variants within the cDNA.", "These differences are non-representative variants within the cDNA. Overall, the BAC sequence differed from the cDNA sequence in 18 positions, 9 of these lead to predicted amino acid substitutions within the polyprotein; one in each of N pro , E rns , E1, E2 and NS3 and four amino acid substitutions in NS5B (Table 1) . When compared to the published reference sequence (GenBank accession AY259122.1), the pBeloR26 BAC sequence differed at an additional 11 positions, 1 of these lead to a predicted amino acid substitution and there was one large insertion (27 nt) in the hypervariable region of the 3′-UTR (Additional file 2: Table S2 ).", "When compared to the published reference sequence (GenBank accession AY259122.1), the pBeloR26 BAC sequence differed at an additional 11 positions, 1 of these lead to a predicted amino acid substitution and there was one large insertion (27 nt) in the hypervariable region of the 3′-UTR (Additional file 2: Table S2 ). To determine the utility of the targeted recombinationmediated mutagenesis system for pestiviruses, two different modifications of the E2 protein coding sequence within pBeloR26 were generated using the Red/ET recombination methodology. Initially, the sequence encoding the linear TAV-epitope (TAVSPTTLR) within the CSFV-E2 was substituted with the sequence encoding the corresponding region (encoding TTVSTSTLA) from the BDV strain \"Gifhorn\" as described in the Materials and Methods section.", "Initially, the sequence encoding the linear TAV-epitope (TAVSPTTLR) within the CSFV-E2 was substituted with the sequence encoding the corresponding region (encoding TTVSTSTLA) from the BDV strain \"Gifhorn\" as described in the Materials and Methods section. More than 90% of the colonies obtained using this procedure contained the required BAC\n\nAnti-CSFV E2 (WH211) Figure 2 Antibody reaction patterns of pestivirus infected cells. SFT-R cells were infected with vR26 and its two derivatives vR26_E2gif and vR26_TAV plus vGifhorn [26] .", "SFT-R cells were infected with vR26 and its two derivatives vR26_E2gif and vR26_TAV plus vGifhorn [26] . After 72 h, the cells were fixed and stained with monoclonal antibodies against the NS3 protein (WB103/105, left column), the CSFV E2 protein (WH303 and WH211, middle columns) and the BDV E2 protein (WB166, right column) as indicated and viewed using a fluorescence microscope. structure as determined by NotI digestions.", "structure as determined by NotI digestions. The complete genome sequences of the CSFV cDNA within two selected BACs, designated pBeloR26_TAV have been verified (data not shown). In addition, the complete coding sequence (1119 nt) for the CSFV-E2 protein was substituted by the corresponding sequence from BDV \"Gifhorn\".", "In addition, the complete coding sequence (1119 nt) for the CSFV-E2 protein was substituted by the corresponding sequence from BDV \"Gifhorn\". Again more than 90% of the colonies obtained contained the required BAC and the same proportion of correctly recombined BACs was obtained using either 30 nt or 50 nt homology arms. The chimeric BAC was designated, pBeloR26_E2gif and the complete virus genome sequence (cDNA) was verified (data not shown).", "The chimeric BAC was designated, pBeloR26_E2gif and the complete virus genome sequence (cDNA) was verified (data not shown). After electroporation with RNA transcripts derived from either pBeloR26_TAV or pBeloR26_E2gif a large number of CSFV NS3-positive cells could be observed (data not shown) and chimeric virus stocks, termed vR26_TAV and vR26_E2gif, were generated after further passages in cells. Cells infected with these viruses and with the parental vR26 and vGifhorn strains were all stained with mAbs directed against the NS3 protein ( Figure 2 ).", "Cells infected with these viruses and with the parental vR26 and vGifhorn strains were all stained with mAbs directed against the NS3 protein ( Figure 2 ). However, in contrast to the parental vR26 virus, the chimeric viruses rescued from the recombined BACs were not recognized by anti-E2 mAbs specific for the CSFV-E2 proteins ( Figure 2 ) and thus, consistent with their structure, displayed the same antibody reaction pattern as vGifhorn. Two different anti-CSFV E2 mAbs, WH211 and WH303, were used for the staining and the latter has been shown previously to target the TAV-epitope [18] .", "Two different anti-CSFV E2 mAbs, WH211 and WH303, were used for the staining and the latter has been shown previously to target the TAV-epitope [18] . As anticipated, cells infected with either the vGifhorn or with the chimeric vR26_E2gif could be shown to express the \"Gifhorn\" E2 protein using staining with an anti-BDV mAb ( Figure 2 ). The presence of the BDV epitope TTVSTSTLA in vR26_ TAV was insufficient to permit efficient recognition by this anti-BDV mab, although a weak signal was observed in some cells.", "The presence of the BDV epitope TTVSTSTLA in vR26_ TAV was insufficient to permit efficient recognition by this anti-BDV mab, although a weak signal was observed in some cells. The BAC constructs pBeloR26 and pBeloR26_E2gif were analysed for the genetic stability of the cDNA to determine the suitability of the BAC vector for maintaining full-length pestivirus cDNAs. E. coli DH10B cells containing the BACs were passaged 15 times, by overnight growth, and the complete viral cDNAs within the BACs were sequenced after the 1st and the 15th passage.", "E. coli DH10B cells containing the BACs were passaged 15 times, by overnight growth, and the complete viral cDNAs within the BACs were sequenced after the 1st and the 15th passage. No mutations were observed within the 12316 nt virus cDNA sequences after this extensive propagation of the BACs in the bacterial host, indicating a highly stable system for the maintenance of complete pestivirus cDNA sequences. The viruses, vR26 and vR26_E2gif, rescued from their respective BAC constructs, were also tested for their genetic stability within mammalian cells.", "The viruses, vR26 and vR26_E2gif, rescued from their respective BAC constructs, were also tested for their genetic stability within mammalian cells. Linearized BAC DNA was transcribed in vitro and the RNA was electroporated into PK15 cells. Three days after electroporation the cells were stained with the anti-NS3 antibody to detect the presence of replicating virus.", "Three days after electroporation the cells were stained with the anti-NS3 antibody to detect the presence of replicating virus. Samples containing virus positive cells were passaged onto new cells, this process \n\n*Nt position 10665 in vR26/P-12 is reverted from A to G as in the parental cDNA. was repeated for 12 separate passages (each of three days).", "was repeated for 12 separate passages (each of three days). The virus titre (as TCID 50 /ml) was determined for each passage. Passage of the rescued vR26_E2gif chimeric virus in PK15 cells resulted in rapidly decreasing virus titres and was discontinued after the 2nd passage ( Figure 4A ).", "Passage of the rescued vR26_E2gif chimeric virus in PK15 cells resulted in rapidly decreasing virus titres and was discontinued after the 2nd passage ( Figure 4A ). Instead, further passage of this chimeric virus was performed in ovine SFT-R cells (the preferred cell type for BDV) and resulted in much higher titers of the chimeric virus. Virus titers reached more than 10 6 TCID 50 /ml after the 1st passage and remained stable for 12 passages ( Figure 4A ).", "Virus titers reached more than 10 6 TCID 50 /ml after the 1st passage and remained stable for 12 passages ( Figure 4A ). The rescued vR26 was also efficiently propagated on the SFT-R cells but maintained a slightly lower titer than the vR26_E2gif chimeric virus ( Figure 4A ). To check that the viruses retained their antibody reaction properties ( Figure 2 ) after these passages, cells were infected with viruses from the 12th SFT-R cell culture passage (termed vR26/P-12 and vR26_E2gif/P-12) and stained with a polyclonal anti-pestivirus serum and with specific mAbs directed against the CSFV-E2 and BDV-E2 proteins ( Figure 4B ).", "To check that the viruses retained their antibody reaction properties ( Figure 2 ) after these passages, cells were infected with viruses from the 12th SFT-R cell culture passage (termed vR26/P-12 and vR26_E2gif/P-12) and stained with a polyclonal anti-pestivirus serum and with specific mAbs directed against the CSFV-E2 and BDV-E2 proteins ( Figure 4B ). Cells infected with either the vR26/P-12 or the chimeric vR26_E2gif/P-12 were each detected by the polyclonal anti-pestivirus serum as expected. The anti-CSFV-E2 mAb specifically detected cells infected with vR26/P-12 but not cells infected by the chimeric virus containing the BDV-E2 protein (consistent with the results shown in Figure 2 ).", "The anti-CSFV-E2 mAb specifically detected cells infected with vR26/P-12 but not cells infected by the chimeric virus containing the BDV-E2 protein (consistent with the results shown in Figure 2 ). In contrast, the anti-BDV-E2 mAb specifically detected infection by the vR26_E2gif/P-12 and did not recognize cells infected with vR26/P-12. Each result is in accord with the structure of the viruses.", "Each result is in accord with the structure of the viruses. The 4th passage of vR26 (vR26/P-4) displayed a slower growth rate than the virus obtained after 12 passages (see Figure 5A ). It also had a reduced growth rate compared to both the vR26_E2gif/P-4 and vR26_E2gif/P-12.", "It also had a reduced growth rate compared to both the vR26_E2gif/P-4 and vR26_E2gif/P-12. The fulllength sequence of pBeloR26 had revealed ten non-silent mutations compared to the reference sequence (AY25 9122.1) for this virus (Additional file 2: Table S2 ). Any of these mutations could be responsible for the impaired growth acting alone or in concert.", "Any of these mutations could be responsible for the impaired growth acting alone or in concert. For further investigation of this issue, full length cDNAs prepared from vR26/ P-4, vR26/P-12, vR26_E2gif/P-4 and vR26_E2gif/P-12 were deep-sequenced using both the 454 FLX and Ion PGM platforms for comparison and to determine the quasispecies distribution (Additional file 3: Figure S1 and Additional file 4: Figure S2 ). Sequencing data from both platforms revealed that both the vR26/P-12 and vR26_E2gif/P-12 were close to 100% changed at nt position A10665G compared to the BAC clones (resulting in the predicted amino acid substitution D3431G within the NS5B protein, the RNAdependent RNA polymerase, see Figure 5B ).", "Sequencing data from both platforms revealed that both the vR26/P-12 and vR26_E2gif/P-12 were close to 100% changed at nt position A10665G compared to the BAC clones (resulting in the predicted amino acid substitution D3431G within the NS5B protein, the RNAdependent RNA polymerase, see Figure 5B ). This adaptation is a reversion back to the consensus cDNA sequence of the parental vaccine virus, vRiemser (Additional file 2: Table S2 ). Additionally, vR26/P-4 and vR26_E2gif/P-4 already showed evidence for this reversion being present within the population.", "Additionally, vR26/P-4 and vR26_E2gif/P-4 already showed evidence for this reversion being present within the population. For vR26/P-4, the level of reversion was 57%, while for vR26_E2gif/P-4 the extent of change was 73% (see Figure 5B ). In this study, we have established the first BAC containing the full-length cDNA of a CSFV vaccine strain.", "In this study, we have established the first BAC containing the full-length cDNA of a CSFV vaccine strain. The BAC differed from the parental cDNA sequence in 18 positions leading to 9 aa substitutions ( Table 1 ). The method that has been used for the generation of pBeloR26 is based on full genome amplification of cDNA followed by direct cloning to obtain the BACs [11] .", "The method that has been used for the generation of pBeloR26 is based on full genome amplification of cDNA followed by direct cloning to obtain the BACs [11] . This approach results in cDNA clones that reflect the quasispecies composition of the parental viral RNA and thus it is not guaranteed to obtain cDNA clones corresponding to the consensus sequence of the cDNA used. However, it is possible to correct the mutations using the BAC recombination approach if a consensus clone is needed.", "However, it is possible to correct the mutations using the BAC recombination approach if a consensus clone is needed. To demonstrate the utility of the Red/ET mediated recombination method we have generated a series of modified BACs derived from this CSFV full-length cDNA. These include BACs with substitution of the linear TAV-epitope present in the E2 protein and also BACs with substitution of the complete E2 protein with heterologous pestivirus sequences.", "These include BACs with substitution of the linear TAV-epitope present in the E2 protein and also BACs with substitution of the complete E2 protein with heterologous pestivirus sequences. We have also used the same approach for a range of different targeted modifications within CSFV BACs including specific deletions and substitutions in the 5′UTR of CSFV [24] and for insertions of heterologous reporter sequences into CSFV replicons [25] . Using Red/ET recombinationmediated mutagenesis for the targeted design, the work can be expedited and focused, in principal, on any sequence within the viral genome and is not dependent on the use of internal restriction sites.", "Using Red/ET recombinationmediated mutagenesis for the targeted design, the work can be expedited and focused, in principal, on any sequence within the viral genome and is not dependent on the use of internal restriction sites. The results demonstrate that Red/ ET recombination-mediated mutagenesis of pestivirus BAC cDNAs provides a useful tool for advancing the construction of modified pestiviruses. Cells infected with the parental vR26 virus were recognized by the two anti-E2 mAbs (WH211 and WH303) specific for the CSFV-E2 proteins, in contrast cells infected with the modified viruses vR26_TAV and vR26_E2gif, rescued from the recombined BACs, were not detected by these mAbs.", "Cells infected with the parental vR26 virus were recognized by the two anti-E2 mAbs (WH211 and WH303) specific for the CSFV-E2 proteins, in contrast cells infected with the modified viruses vR26_TAV and vR26_E2gif, rescued from the recombined BACs, were not detected by these mAbs. Furthermore, as expected, cells infected with the vR26_E2gif were recognized by the anti-BDV mAb (WB166) whereas no staining was observed with this antibody in vR26 infected cells or in cells with vR26_TAV. The mAb WH303 recognizes the CSFV TAV-epitope [18] and the difference in 4 aa between the TAV-epitope and the corresponding sequence from BDV strain \"Gifhorn\" is enough to completely abolish the recognition by this mAb.", "The mAb WH303 recognizes the CSFV TAV-epitope [18] and the difference in 4 aa between the TAV-epitope and the corresponding sequence from BDV strain \"Gifhorn\" is enough to completely abolish the recognition by this mAb. The lack of staining of vR26_TAV infected cells by the WH211 indicated that the TAV-sequence is also important for the epitope recognized by this mAb. Thus, the chimeric pestiviruses, vR26_TAV and vR26_E2gif, containing heterologous E2 sequences can be readily discriminated from the vR26 using specific anti-E2 monoclonal antibodies.", "Thus, the chimeric pestiviruses, vR26_TAV and vR26_E2gif, containing heterologous E2 sequences can be readily discriminated from the vR26 using specific anti-E2 monoclonal antibodies. These new chimeric pestiviruses represents Cstrain based marked vaccine candidates with the characteristics desired for safe and efficacious DIVA vaccines against CSFV. Indeed, vR26_E2gif vaccinated pigs could be efficiently discriminated from C-strain vaccinated pigs and from CSFV infected pigs using CSFV-E2 specific antibody ELISAs (Rasmussen et al., unpublished results).", "Indeed, vR26_E2gif vaccinated pigs could be efficiently discriminated from C-strain vaccinated pigs and from CSFV infected pigs using CSFV-E2 specific antibody ELISAs (Rasmussen et al., unpublished results). Nucleotide sequence data for the pBeloR26 showed a number of changes from the published reference sequence for \"C-strain Riems\". Some of these differences are present in the cDNA derived from the vaccine stock at a detectable level whereas others may represent low-level variants within the cDNA or errors introduced by the RT-PCR amplification.", "Some of these differences are present in the cDNA derived from the vaccine stock at a detectable level whereas others may represent low-level variants within the cDNA or errors introduced by the RT-PCR amplification. Full-length sequencing revealed that no changes occurred in the cDNA during extensive propagation in E. coli DH10B of the pBeloR26 and the E2chimeric derivative, pBeloR26_E2gif, indicating a very high stability of these BAC-cloned CSFV cDNAs. This is essential if this system is to be useful for cloning and sequence manipulation, and contrasts with stability problems encountered with conventional plasmids containing fulllength pestivirus cDNAs [31] .", "This is essential if this system is to be useful for cloning and sequence manipulation, and contrasts with stability problems encountered with conventional plasmids containing fulllength pestivirus cDNAs [31] . The stability of these BACs is consistent with previous reports on the stability of BACs containing other viruses of the family Flaviviridae in E. coli [8, 10] . Extensive passaging of the rescued vR26 and the chimeric virus derivative, vR26_E2gif, resulted in a change at nucleotide position A10665G (resulting in the predicted aa" ]

[ "BACKGROUND: Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria.", "This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. RESULTS: A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria.", "Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that modifications in the E2 protein coding sequence were stably maintained. A single amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence.", "A single amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence. CONCLUSIONS: These results show that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses. Text: Bacterial artificial chromosomes (BACs) are ideally suited for the stable maintenance of large DNA sequences derived from viral genomes [1] .", "Text: Bacterial artificial chromosomes (BACs) are ideally suited for the stable maintenance of large DNA sequences derived from viral genomes [1] . A considerable number of BAC systems have been established for large DNA viruses; in particular many different herpesvirus genomes have been cloned into BACs (for review see [2] ). The first BAC systems using RNA virus cDNAs were described for coronaviruses [3] [4] [5] [6] and recently the first BAC containing a full-length cDNA for a negative-stranded RNA virus was described [7] .", "The first BAC systems using RNA virus cDNAs were described for coronaviruses [3] [4] [5] [6] and recently the first BAC containing a full-length cDNA for a negative-stranded RNA virus was described [7] . Similarly, cDNAs corresponding to the full-length genomes of members of the Flaviviridae family (Japanese encephalitis virus [8] and Dengue virus [9] ) have been inserted into BACs. BACs containing full-length cDNAs of pestiviruses (also within the Flaviviridae), including bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) have recently been established [10, 11] .", "BACs containing full-length cDNAs of pestiviruses (also within the Flaviviridae), including bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) have recently been established [10, 11] . Infectious pestiviruses can be rescued using RNA transcripts derived from these BACs. The pestiviruses have single stranded positive sense RNA genomes, about 12.3 kb in length, which includes a single long open reading frame, encoding a large polyprotein, flanked by 5′ and 3′ untranslated regions (UTRs) that are critical for autonomous replication of the genome [12, 13] .", "The pestiviruses have single stranded positive sense RNA genomes, about 12.3 kb in length, which includes a single long open reading frame, encoding a large polyprotein, flanked by 5′ and 3′ untranslated regions (UTRs) that are critical for autonomous replication of the genome [12, 13] . The polyprotein is cleaved by cellular and viral proteases into four structural proteins (nucleocapsid protein C, envelope glycoproteins E rns , E1 and E2) and eight nonstructural proteins (N pro , p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). The availability of genetically defined and stable pestivirus BACs facilitates the functional study of viral proteins or RNA structures and also the development of new marker vaccine candidates.", "The availability of genetically defined and stable pestivirus BACs facilitates the functional study of viral proteins or RNA structures and also the development of new marker vaccine candidates. Several CSFV vaccines with marker properties based on chimeric pestiviruses have been developed over the years [14] . In particular, chimeric pestiviruses with substitution of the entire E2 protein have been described [15] [16] [17] but also mutants with more subtle modifications, such as the modification of the important TAV-epitope [18] within the CSFV-E2 protein [19, 20] are promising marker vaccine candidates.", "In particular, chimeric pestiviruses with substitution of the entire E2 protein have been described [15] [16] [17] but also mutants with more subtle modifications, such as the modification of the important TAV-epitope [18] within the CSFV-E2 protein [19, 20] are promising marker vaccine candidates. Manipulation of BACs using traditional cloning procedures can be difficult (e.g. because of a lack of convenient restriction enzyme sites) and thus a range of methodologies that apply bacterial genetics, including homologous recombination (e.g.", "because of a lack of convenient restriction enzyme sites) and thus a range of methodologies that apply bacterial genetics, including homologous recombination (e.g. Red/ET homologous recombineering) within the E. coli host, have been developed (for review, see [21] ). The use of homologous recombination allows site-directed mutagenesis of BACs [22] and, by employing a counterselection scheme, specific modifications can be obtained without leaving residual \"foreign\" sequences [23] .", "The use of homologous recombination allows site-directed mutagenesis of BACs [22] and, by employing a counterselection scheme, specific modifications can be obtained without leaving residual \"foreign\" sequences [23] . The main advantage of this method is that there are no target limitations (e.g. based on size or location) and no need for suitable restriction sites.", "based on size or location) and no need for suitable restriction sites. The integration of the modified sequence is performed in vivo (within E. coli) thereby potentially being more accurate than in vitro approaches like PCR-based methods. Although in vitro cloning approaches based on the use of high-fidelity polymerases for PCR amplification have significantly improved in recent years, the use of in vivo approaches should allow a more accurate method of mutagenesis due to the use of the cells own high-fidelity replication system which includes proof reading.", "Although in vitro cloning approaches based on the use of high-fidelity polymerases for PCR amplification have significantly improved in recent years, the use of in vivo approaches should allow a more accurate method of mutagenesis due to the use of the cells own high-fidelity replication system which includes proof reading. Whereas BAC recombination has been commonly used for modifying DNA viruses, there are only very few reports about the use of this technology for RNA viruses [7, 24, 25] . Here, a generally applicable strategy for the manipulation and rescue of chimeric pestiviruses from BACs is described as a model, and the flexibility of this approach is demonstrated by generating different modifications in the viral cDNA of the new CSFV-BAC, pBeloR26, derived from the modified live vaccine strain \"C-strain Riems\".", "Here, a generally applicable strategy for the manipulation and rescue of chimeric pestiviruses from BACs is described as a model, and the flexibility of this approach is demonstrated by generating different modifications in the viral cDNA of the new CSFV-BAC, pBeloR26, derived from the modified live vaccine strain \"C-strain Riems\". The targeted recombination-mediated mutagenesis described here includes the substitution of the 9 amino acid (aa) linear TAV-epitope (TAVSPTTLR) present in the E2 protein with the corresponding region (TTVSTSTLA) of a heterologous pestivirus (border disease virus, BDV, strain \"Gifhorn\") and also the replacement of the entire CSFV E2 protein coding region with the whole E2 coding region from the same BDV, to generate marked vaccine viruses that can be discriminated using specific anti-E2 monoclonal antibodies. The genetic stabilities of both the BAC constructs (within E. coli) and the rescued viruses have also been assessed.", "The genetic stabilities of both the BAC constructs (within E. coli) and the rescued viruses have also been assessed. Porcine kidney (PK15) and sheep fetal thymoid (SFT-R) cells were grown at 37°C (with 5% (v/v) CO 2 ) in Dulbecco's minimal essential medium (DMEM) supplemented with 5% (v/v) pestivirus-free fetal calf serum. Virus from a bait containing the modified live vaccine CSFV \"C-strain Riems\" (Riemser Arzneimittel AG, Germany) was propagated once in PK15 cells and termed vRiemser.", "Virus from a bait containing the modified live vaccine CSFV \"C-strain Riems\" (Riemser Arzneimittel AG, Germany) was propagated once in PK15 cells and termed vRiemser. RNA obtained from BDV strain \"Gifhorn\" [26] was used for amplification of the Gifhorn E2-coding sequence. Oligonucleotide primers used are listed in Additional file 1: Table S1 .", "Oligonucleotide primers used are listed in Additional file 1: Table S1 . The BAC construct, pBeloR26, was constructed using the long RT-PCR method as previously described [11] using RNA derived from the \"C-strain Riems\". Briefly, full-length viral cDNAs flanked by NotI sites were amplified by long RT-PCR using primers 5′Cstrain_T7_Not1 (which includes a T7 promotor for in vitro transcription, a NotI site and a region corresponding to the first 44 nt of the genome) and 3′CSFV_Not1 (that contains a NotI site and sequence complementary to the 3′-terminal 35 nt of the genome that are conserved among many CSFVs including the Cstrain).", "Briefly, full-length viral cDNAs flanked by NotI sites were amplified by long RT-PCR using primers 5′Cstrain_T7_Not1 (which includes a T7 promotor for in vitro transcription, a NotI site and a region corresponding to the first 44 nt of the genome) and 3′CSFV_Not1 (that contains a NotI site and sequence complementary to the 3′-terminal 35 nt of the genome that are conserved among many CSFVs including the Cstrain). The product (ca. 12.3 kbp) was digested with NotI and inserted into similarly digested pBeloBAC11 (New England Biolabs, GenBank accession U51113).", "12.3 kbp) was digested with NotI and inserted into similarly digested pBeloBAC11 (New England Biolabs, GenBank accession U51113). All BACs were modified and maintained in E. coli DH10B cells (Invitrogen) grown at 37°C in LB medium containing chloramphenicol (Cam, 15 μg/ml). The electroporation of bacteria was performed in 0.1 cm cuvettes using 1 pulse at 1800 V, 25 μF and 200 Ω in a Gene Pulser Xcell (Bio-Rad).", "The electroporation of bacteria was performed in 0.1 cm cuvettes using 1 pulse at 1800 V, 25 μF and 200 Ω in a Gene Pulser Xcell (Bio-Rad). BACs to be used as templates for long PCR or for screening by restriction enzyme digestion were purified from 4 ml overnight cultures of E. coli DH10B using the ZR BAC DNA Miniprep Kit (Zymo Research). BACs required for direct genome sequencing were purified from 500 ml cultures using the Large-construct kit (Qiagen).", "BACs required for direct genome sequencing were purified from 500 ml cultures using the Large-construct kit (Qiagen). Modifications to the full-length CSFV cDNA were accomplished in E. coli DH10B (streptomycin resistant, Strep R ) using the Counter Selection BAC Modification Kit (Gene Bridges, Heidelberg, Germany). The Red/ET recombination involved three steps (i-iii).", "The Red/ET recombination involved three steps (i-iii). Step i) the temperature-sensitive pRedET expression plasmid (Gene Bridges) was introduced into electroporationcompetent E.coli DH10B cells containing the parental BAC (phenotype Cam R , Strep R ). The pRedET expresses the phage lambda proteins redα, redβ and redγ, under control of the arabinose-inducible pBAD promoter, allowing homologous recombination to occur.", "The pRedET expresses the phage lambda proteins redα, redβ and redγ, under control of the arabinose-inducible pBAD promoter, allowing homologous recombination to occur. Immediately after electroporation, pre-warmed LB medium without antibiotics (1 ml) was added to the cells which were then incubated at 30°C for 1 hour, prior to spreading onto agar plates containing Cam (15 μg/ml) and tetracycline (Tet) (3 μg/ml) and then incubated at 30°C overnight to maintain the pRedET. The presence of the pRedET plasmid (conferring Tet R ) was verified by visual inspection of BAC-DNA preparations from the Cam R /Tet R colonies using agarose gel electrophoresis.", "The presence of the pRedET plasmid (conferring Tet R ) was verified by visual inspection of BAC-DNA preparations from the Cam R /Tet R colonies using agarose gel electrophoresis. Step ii) counter-selection marker cassettes with an extra NotI site for screening purposes (rpsL-neo, 1325 bp) were amplified by PCR using primers with 30 nt or 50 nt extensions that were homologous to the target site in the BAC using the rpsL-neo plasmid (Gene Bridges) as template and the Phusion hot start II HF DNA polymerase (Thermo Scientific) with cycling conditions as follows: 98°C for 30s, followed by 35 cycles of 98°C for 10s, 60°C for 20s, 72°C for 60s, and 1 cycle at 72°C for 4 min. The PCR products (ca.", "The PCR products (ca. 1400 bp) were isolated on 1% (w/v) TBE agarose gels and purified using a GeneJET gel extraction kit (Thermo Scientific). Samples (30 μl), from an E. coli culture containing pRedET and the parental BAC grown overnight at 30°C in LB media (Cam, Tet), were used to inoculate 1.4 ml of fresh LB media with the same antibiotics to obtain exponentially growing bacteria at 30°C.", "Samples (30 μl), from an E. coli culture containing pRedET and the parental BAC grown overnight at 30°C in LB media (Cam, Tet), were used to inoculate 1.4 ml of fresh LB media with the same antibiotics to obtain exponentially growing bacteria at 30°C. Red/ET recombination proteins were induced by adding 50 μl of 10% (w/v) L-arabinose (Sigma). The PCR product (200 ng) containing the rpsL-neo cassette was introduced into these bacteria using electroporation (as above).", "The PCR product (200 ng) containing the rpsL-neo cassette was introduced into these bacteria using electroporation (as above). Following electroporation, the cells were grown at 37°C for 70 min (to allow recombination) and then selected on plates containing Cam (15 μg/ml), Tet (3 μg/ml) and kanamycin (Kan, 15 μg/ml) overnight at 30°C to maintain the pRedET. Note, the rpsL cassette confers Streptomycin sensitivity (Strep S ) onto the resistant DH10B strain and the neo confers Kanamycin resistance (Kan R ).", "Note, the rpsL cassette confers Streptomycin sensitivity (Strep S ) onto the resistant DH10B strain and the neo confers Kanamycin resistance (Kan R ). The correct phenotype (Cam R , Kan R , Tet R , Strep S ) of the resulting colonies was confirmed by streaking the colonies onto plates containing Cam (15 μg/ml), Tet (3 μg/ml) and Kan (15 μg/ml) and grown at 30°C. Importantly, for the third step, the replacement of the rpsL-neo cassette (using counter-selection), the selected colonies were also streaked onto plates containing Cam (15 μg/ml) plus Strep (50 μg/ml) and shown to be Strep S indicating incorporation of a functional rpsL gene.", "Importantly, for the third step, the replacement of the rpsL-neo cassette (using counter-selection), the selected colonies were also streaked onto plates containing Cam (15 μg/ml) plus Strep (50 μg/ml) and shown to be Strep S indicating incorporation of a functional rpsL gene. The structures of the intermediate BACs were verified by restriction enzyme analysis and sequencing around the inserts. Step iii) the replacement of the rpsL-neo selection cassettes from the intermediate constructs using linear DNA fragments was achieved through counter-selection and Red/ET recombination.", "Step iii) the replacement of the rpsL-neo selection cassettes from the intermediate constructs using linear DNA fragments was achieved through counter-selection and Red/ET recombination. Again, the homologous sequences at the ends of the DNA fragment were used for Red/ET mediated recombination events to replace the rpsL-neo cassette with the sequence of interest. Counterselection against the rpsL-neo cassette (phenotype Cam R , Kan R , Tet R , Strep S ) was employed using media containing Cam (15 μg/ml) and Strep (50 μg/ml) to isolate the required derivatives (phenotype Cam R and Strep R ).", "Counterselection against the rpsL-neo cassette (phenotype Cam R , Kan R , Tet R , Strep S ) was employed using media containing Cam (15 μg/ml) and Strep (50 μg/ml) to isolate the required derivatives (phenotype Cam R and Strep R ). Initially, the intermediate construct, pBeloR26_E2rpsLneo ( Figure 1 ), was generated using Red/ET recombination by insertion of the rpsL-neo cassette with an extra NotI site for screening purposes which was amplified using primers Criems-TAVfor and Criems-TAVrev (Additional file 1: Table S1 ) in place of the TAVSPTTLR coding sequence (27 nt) . Secondly, the rpsL-neo cassette in this intermediate construct was then replaced using counter-selection Red/ ET recombination using a single-stranded oligonucleotide, Riems_TAV_Gifhorn (Additional file 1: Table S1 ) with the same homology arms as used for the rpsL-neo cassette, to introduce the coding sequence for the BDV \"Gifhorn\" epitope sequence (TTVSTSTLA).", "Secondly, the rpsL-neo cassette in this intermediate construct was then replaced using counter-selection Red/ ET recombination using a single-stranded oligonucleotide, Riems_TAV_Gifhorn (Additional file 1: Table S1 ) with the same homology arms as used for the rpsL-neo cassette, to introduce the coding sequence for the BDV \"Gifhorn\" epitope sequence (TTVSTSTLA). The resulting construct was named pBeloR26_TAV (Figure 1 ). The initial intermediate construct (with rpsL-neo) was then used to produce the pBeloR26_E2gif construct ( Figure 1 ).", "The initial intermediate construct (with rpsL-neo) was then used to produce the pBeloR26_E2gif construct ( Figure 1 ). For this, the E2 coding sequence was amplified from cDNA prepared from BDV \"Gifhorn\" RNA using two different primer pairs, one set with 50 nt homology arms (Criems_E2_gifFlong/Criems_ E2_gifRlong) and another with 30 nt homologous sequences (Criems_E2_gifF/Criems_E2_gifR). For generation of BACs with substitution of the entire E2 coding sequences, PCR products consisting of the sequence of interest flanked with homology arms identical to the target area were generated by PCR (as for the rpsLneo cassette).", "For generation of BACs with substitution of the entire E2 coding sequences, PCR products consisting of the sequence of interest flanked with homology arms identical to the target area were generated by PCR (as for the rpsLneo cassette). For making constructs with substitution of shorter sequences (e.g. the TAV-epitope), the recombination was achieved using synthetic single stranded oligonucleotides rather than PCR products.", "the TAV-epitope), the recombination was achieved using synthetic single stranded oligonucleotides rather than PCR products. Pre-heating of single stranded oligonucleotides at 95°C for 2 min followed by snap-freezing, prior to electroporation, empirically showed the best results. In each case, the DNA molecules were introduced into E. coli containing the BAC derivatives including the rpsL-neo cassettes together with the pRedET plasmid by electroporation as described above.", "In each case, the DNA molecules were introduced into E. coli containing the BAC derivatives including the rpsL-neo cassettes together with the pRedET plasmid by electroporation as described above. The structures of the modified BACs were verified by restriction enzyme analysis and subsequent full-genome sequencing (see below). BAC DNA (1 μg) was linearized with NotI or 1 μl BAC DNA was used as template for long PCR amplification using primers 5′C-strain_T7_Not1 and 3′CSFV (Additional file 1: Table S1 ).", "BAC DNA (1 μg) was linearized with NotI or 1 μl BAC DNA was used as template for long PCR amplification using primers 5′C-strain_T7_Not1 and 3′CSFV (Additional file 1: Table S1 ). Linearized BACs or PCR products were purified with the GeneJet PCR purification kit (Thermo Scientific) and transcribed in vitro using a Megascript T7 kit (Invitrogen). Viruses were rescued from RNA transcripts (1 to 5 μg) by electroporation of porcine (PK15) or ovine (SFT-R) cells essentially as described previously [24] .", "Viruses were rescued from RNA transcripts (1 to 5 μg) by electroporation of porcine (PK15) or ovine (SFT-R) cells essentially as described previously [24] . Cells were analysed using immunofluorescence microscopy (typically after 3 days) for the expression of NS3 and E2 proteins using specific monoclonal antibodies (mAbs), these were anti-NS3 (WB103/105, pan-pestivirus), anti-CSFV E2 (WH211, WH303, both CSFV specific) and anti-BDV E2 (WB166, BVDV/BDV specific) (AHVLA Scientific, United Kingdom) together with Alexa 488 conjugated goat antimouse IgG antibody (Molecular Probes, Invitrogen). The nuclei of cells were visualized using DAPI (Vector Laboratories) and images were recorded using a BX63 fluorescence microscope (Olympus).", "The nuclei of cells were visualized using DAPI (Vector Laboratories) and images were recorded using a BX63 fluorescence microscope (Olympus). For peroxidase staining, cells were fixed and stained for the presence of pestivirus antigens using biotinylated pig anti-CSFV/BVDV polyclonal IgG followed by avidin-conjugated horseradish peroxidase (eBioscience) as previously described [27] . The same staining procedure was also performed using the anti-E2 mAbs.", "The same staining procedure was also performed using the anti-E2 mAbs. Samples containing virus-positive cells were passaged onto new cells. Virus growth curves were generated as previously described [24] . Briefly, PK15 or SFT-R cells were infected at a multiplicity of infection (MOI) of 0.1 pfu/cell and grown for three days.", "Briefly, PK15 or SFT-R cells were infected at a multiplicity of infection (MOI) of 0.1 pfu/cell and grown for three days. BAC DNAs (5 μg), purified using the Large-construct kit (Qiagen), or PCR products (1 μg) amplified from viral cDNA or from BACs using the long PCR method (as above) were consensus sequenced using a 454 FLX (Roche) or an Ion PGM (Life Technologies). Both Newbler (Roche) and the bwa.bwasw alignment algorithm [28] were used for mapping the reads to the expected sequence.", "Both Newbler (Roche) and the bwa.bwasw alignment algorithm [28] were used for mapping the reads to the expected sequence. A combination of Samtools [29] and LoFreq SNV-caller [30] was used for downstream single nucleotide variant (SNV) analysis. Finally, clone consensus sequences were aligned using MAFFT in the Geneious software platform (Biomatters).", "Finally, clone consensus sequences were aligned using MAFFT in the Geneious software platform (Biomatters). Generation of a BAC containing full-length cDNA corresponding to the modified live vaccine \"C-strain Riems\"\n\nBACs containing the full-length cDNA corresponding to the parental vRiemser (\"C-strain Riems\") were constructed according to the method described previously for the \"Paderborn\" strain of CSFV [11] . BACs containing the complete CSFV cDNAs were identified by restriction Figure 1 Schematic representation of the CSFV genome organization and the BACs constructed and used in this study.", "BACs containing the complete CSFV cDNAs were identified by restriction Figure 1 Schematic representation of the CSFV genome organization and the BACs constructed and used in this study. Nucleotide (nt) and amino acid (aa) positions within R26 for the 5′ and 3′ termini together with the translational start and stop codons of the polyprotein coding region plus cleavage sites used to make the individual proteins (N pro , C, E rns , E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) are indicated. Insertion of the rpsL-neo in place of the TAV-epitope within CSFV E2 for the intermediate construct (R26_rpsLneo) and the subsequent replacement with the TTVSTSTLA sequence (R26_TAV) and the complete substitution of the E2 sequence (R26_E2gif) are shown.", "Insertion of the rpsL-neo in place of the TAV-epitope within CSFV E2 for the intermediate construct (R26_rpsLneo) and the subsequent replacement with the TTVSTSTLA sequence (R26_TAV) and the complete substitution of the E2 sequence (R26_E2gif) are shown. Names of BAC constructs begin with \"pBelo\" and rescued viruses with \"v\" (e.g. pBeloR26 and vR26).", "pBeloR26 and vR26). Cell culture passage no. of virus is indicated with \"/P\" (e.g. vR26/P-4). digest analysis and following linearization by NotI, RNA transcripts were produced and electroporated into PK15 cells.", "digest analysis and following linearization by NotI, RNA transcripts were produced and electroporated into PK15 cells. This screening resulted in the identification of a BAC containing a cDNA insert of 12316 nt, pBeloR26 (Figure 1) , which yielded infectious virus, termed vR26, that could be propagated in SFT-R cells (Figure 2 , upper panels) and in PK15 cells (Figure 3 ). The rescued vR26 displayed higher growth rate at the early stage (about 10fold difference in virus yield at 24 h) compared to the parental vaccine virus, but after 48 hours similar virus titres were obtained (Figure 3 ).", "The rescued vR26 displayed higher growth rate at the early stage (about 10fold difference in virus yield at 24 h) compared to the parental vaccine virus, but after 48 hours similar virus titres were obtained (Figure 3 ). Full-genome sequencing of the cloned BAC template, pBeloR26, revealed a number of differences throughout the genome when compared to the full-length consensus sequence of the cDNA used for the cloning procedure (see Table 1 ). These differences are non-representative variants within the cDNA.", "These differences are non-representative variants within the cDNA. Overall, the BAC sequence differed from the cDNA sequence in 18 positions, 9 of these lead to predicted amino acid substitutions within the polyprotein; one in each of N pro , E rns , E1, E2 and NS3 and four amino acid substitutions in NS5B (Table 1) . When compared to the published reference sequence (GenBank accession AY259122.1), the pBeloR26 BAC sequence differed at an additional 11 positions, 1 of these lead to a predicted amino acid substitution and there was one large insertion (27 nt) in the hypervariable region of the 3′-UTR (Additional file 2: Table S2 ).", "When compared to the published reference sequence (GenBank accession AY259122.1), the pBeloR26 BAC sequence differed at an additional 11 positions, 1 of these lead to a predicted amino acid substitution and there was one large insertion (27 nt) in the hypervariable region of the 3′-UTR (Additional file 2: Table S2 ). To determine the utility of the targeted recombinationmediated mutagenesis system for pestiviruses, two different modifications of the E2 protein coding sequence within pBeloR26 were generated using the Red/ET recombination methodology. Initially, the sequence encoding the linear TAV-epitope (TAVSPTTLR) within the CSFV-E2 was substituted with the sequence encoding the corresponding region (encoding TTVSTSTLA) from the BDV strain \"Gifhorn\" as described in the Materials and Methods section.", "Initially, the sequence encoding the linear TAV-epitope (TAVSPTTLR) within the CSFV-E2 was substituted with the sequence encoding the corresponding region (encoding TTVSTSTLA) from the BDV strain \"Gifhorn\" as described in the Materials and Methods section. More than 90% of the colonies obtained using this procedure contained the required BAC\n\nAnti-CSFV E2 (WH211) Figure 2 Antibody reaction patterns of pestivirus infected cells. SFT-R cells were infected with vR26 and its two derivatives vR26_E2gif and vR26_TAV plus vGifhorn [26] .", "SFT-R cells were infected with vR26 and its two derivatives vR26_E2gif and vR26_TAV plus vGifhorn [26] . After 72 h, the cells were fixed and stained with monoclonal antibodies against the NS3 protein (WB103/105, left column), the CSFV E2 protein (WH303 and WH211, middle columns) and the BDV E2 protein (WB166, right column) as indicated and viewed using a fluorescence microscope. structure as determined by NotI digestions.", "structure as determined by NotI digestions. The complete genome sequences of the CSFV cDNA within two selected BACs, designated pBeloR26_TAV have been verified (data not shown). In addition, the complete coding sequence (1119 nt) for the CSFV-E2 protein was substituted by the corresponding sequence from BDV \"Gifhorn\".", "In addition, the complete coding sequence (1119 nt) for the CSFV-E2 protein was substituted by the corresponding sequence from BDV \"Gifhorn\". Again more than 90% of the colonies obtained contained the required BAC and the same proportion of correctly recombined BACs was obtained using either 30 nt or 50 nt homology arms. The chimeric BAC was designated, pBeloR26_E2gif and the complete virus genome sequence (cDNA) was verified (data not shown).", "The chimeric BAC was designated, pBeloR26_E2gif and the complete virus genome sequence (cDNA) was verified (data not shown). After electroporation with RNA transcripts derived from either pBeloR26_TAV or pBeloR26_E2gif a large number of CSFV NS3-positive cells could be observed (data not shown) and chimeric virus stocks, termed vR26_TAV and vR26_E2gif, were generated after further passages in cells. Cells infected with these viruses and with the parental vR26 and vGifhorn strains were all stained with mAbs directed against the NS3 protein ( Figure 2 ).", "Cells infected with these viruses and with the parental vR26 and vGifhorn strains were all stained with mAbs directed against the NS3 protein ( Figure 2 ). However, in contrast to the parental vR26 virus, the chimeric viruses rescued from the recombined BACs were not recognized by anti-E2 mAbs specific for the CSFV-E2 proteins ( Figure 2 ) and thus, consistent with their structure, displayed the same antibody reaction pattern as vGifhorn. Two different anti-CSFV E2 mAbs, WH211 and WH303, were used for the staining and the latter has been shown previously to target the TAV-epitope [18] .", "Two different anti-CSFV E2 mAbs, WH211 and WH303, were used for the staining and the latter has been shown previously to target the TAV-epitope [18] . As anticipated, cells infected with either the vGifhorn or with the chimeric vR26_E2gif could be shown to express the \"Gifhorn\" E2 protein using staining with an anti-BDV mAb ( Figure 2 ). The presence of the BDV epitope TTVSTSTLA in vR26_ TAV was insufficient to permit efficient recognition by this anti-BDV mab, although a weak signal was observed in some cells.", "The presence of the BDV epitope TTVSTSTLA in vR26_ TAV was insufficient to permit efficient recognition by this anti-BDV mab, although a weak signal was observed in some cells. The BAC constructs pBeloR26 and pBeloR26_E2gif were analysed for the genetic stability of the cDNA to determine the suitability of the BAC vector for maintaining full-length pestivirus cDNAs. E. coli DH10B cells containing the BACs were passaged 15 times, by overnight growth, and the complete viral cDNAs within the BACs were sequenced after the 1st and the 15th passage.", "E. coli DH10B cells containing the BACs were passaged 15 times, by overnight growth, and the complete viral cDNAs within the BACs were sequenced after the 1st and the 15th passage. No mutations were observed within the 12316 nt virus cDNA sequences after this extensive propagation of the BACs in the bacterial host, indicating a highly stable system for the maintenance of complete pestivirus cDNA sequences. The viruses, vR26 and vR26_E2gif, rescued from their respective BAC constructs, were also tested for their genetic stability within mammalian cells.", "The viruses, vR26 and vR26_E2gif, rescued from their respective BAC constructs, were also tested for their genetic stability within mammalian cells. Linearized BAC DNA was transcribed in vitro and the RNA was electroporated into PK15 cells. Three days after electroporation the cells were stained with the anti-NS3 antibody to detect the presence of replicating virus.", "Three days after electroporation the cells were stained with the anti-NS3 antibody to detect the presence of replicating virus. Samples containing virus positive cells were passaged onto new cells, this process \n\n*Nt position 10665 in vR26/P-12 is reverted from A to G as in the parental cDNA. was repeated for 12 separate passages (each of three days).", "was repeated for 12 separate passages (each of three days). The virus titre (as TCID 50 /ml) was determined for each passage. Passage of the rescued vR26_E2gif chimeric virus in PK15 cells resulted in rapidly decreasing virus titres and was discontinued after the 2nd passage ( Figure 4A ).", "Passage of the rescued vR26_E2gif chimeric virus in PK15 cells resulted in rapidly decreasing virus titres and was discontinued after the 2nd passage ( Figure 4A ). Instead, further passage of this chimeric virus was performed in ovine SFT-R cells (the preferred cell type for BDV) and resulted in much higher titers of the chimeric virus. Virus titers reached more than 10 6 TCID 50 /ml after the 1st passage and remained stable for 12 passages ( Figure 4A ).", "Virus titers reached more than 10 6 TCID 50 /ml after the 1st passage and remained stable for 12 passages ( Figure 4A ). The rescued vR26 was also efficiently propagated on the SFT-R cells but maintained a slightly lower titer than the vR26_E2gif chimeric virus ( Figure 4A ). To check that the viruses retained their antibody reaction properties ( Figure 2 ) after these passages, cells were infected with viruses from the 12th SFT-R cell culture passage (termed vR26/P-12 and vR26_E2gif/P-12) and stained with a polyclonal anti-pestivirus serum and with specific mAbs directed against the CSFV-E2 and BDV-E2 proteins ( Figure 4B ).", "To check that the viruses retained their antibody reaction properties ( Figure 2 ) after these passages, cells were infected with viruses from the 12th SFT-R cell culture passage (termed vR26/P-12 and vR26_E2gif/P-12) and stained with a polyclonal anti-pestivirus serum and with specific mAbs directed against the CSFV-E2 and BDV-E2 proteins ( Figure 4B ). Cells infected with either the vR26/P-12 or the chimeric vR26_E2gif/P-12 were each detected by the polyclonal anti-pestivirus serum as expected. The anti-CSFV-E2 mAb specifically detected cells infected with vR26/P-12 but not cells infected by the chimeric virus containing the BDV-E2 protein (consistent with the results shown in Figure 2 ).", "The anti-CSFV-E2 mAb specifically detected cells infected with vR26/P-12 but not cells infected by the chimeric virus containing the BDV-E2 protein (consistent with the results shown in Figure 2 ). In contrast, the anti-BDV-E2 mAb specifically detected infection by the vR26_E2gif/P-12 and did not recognize cells infected with vR26/P-12. Each result is in accord with the structure of the viruses.", "Each result is in accord with the structure of the viruses. The 4th passage of vR26 (vR26/P-4) displayed a slower growth rate than the virus obtained after 12 passages (see Figure 5A ). It also had a reduced growth rate compared to both the vR26_E2gif/P-4 and vR26_E2gif/P-12.", "It also had a reduced growth rate compared to both the vR26_E2gif/P-4 and vR26_E2gif/P-12. The fulllength sequence of pBeloR26 had revealed ten non-silent mutations compared to the reference sequence (AY25 9122.1) for this virus (Additional file 2: Table S2 ). Any of these mutations could be responsible for the impaired growth acting alone or in concert.", "Any of these mutations could be responsible for the impaired growth acting alone or in concert. For further investigation of this issue, full length cDNAs prepared from vR26/ P-4, vR26/P-12, vR26_E2gif/P-4 and vR26_E2gif/P-12 were deep-sequenced using both the 454 FLX and Ion PGM platforms for comparison and to determine the quasispecies distribution (Additional file 3: Figure S1 and Additional file 4: Figure S2 ). Sequencing data from both platforms revealed that both the vR26/P-12 and vR26_E2gif/P-12 were close to 100% changed at nt position A10665G compared to the BAC clones (resulting in the predicted amino acid substitution D3431G within the NS5B protein, the RNAdependent RNA polymerase, see Figure 5B ).", "Sequencing data from both platforms revealed that both the vR26/P-12 and vR26_E2gif/P-12 were close to 100% changed at nt position A10665G compared to the BAC clones (resulting in the predicted amino acid substitution D3431G within the NS5B protein, the RNAdependent RNA polymerase, see Figure 5B ). This adaptation is a reversion back to the consensus cDNA sequence of the parental vaccine virus, vRiemser (Additional file 2: Table S2 ). Additionally, vR26/P-4 and vR26_E2gif/P-4 already showed evidence for this reversion being present within the population.", "Additionally, vR26/P-4 and vR26_E2gif/P-4 already showed evidence for this reversion being present within the population. For vR26/P-4, the level of reversion was 57%, while for vR26_E2gif/P-4 the extent of change was 73% (see Figure 5B ). In this study, we have established the first BAC containing the full-length cDNA of a CSFV vaccine strain.", "In this study, we have established the first BAC containing the full-length cDNA of a CSFV vaccine strain. The BAC differed from the parental cDNA sequence in 18 positions leading to 9 aa substitutions ( Table 1 ). The method that has been used for the generation of pBeloR26 is based on full genome amplification of cDNA followed by direct cloning to obtain the BACs [11] .", "The method that has been used for the generation of pBeloR26 is based on full genome amplification of cDNA followed by direct cloning to obtain the BACs [11] . This approach results in cDNA clones that reflect the quasispecies composition of the parental viral RNA and thus it is not guaranteed to obtain cDNA clones corresponding to the consensus sequence of the cDNA used. However, it is possible to correct the mutations using the BAC recombination approach if a consensus clone is needed.", "However, it is possible to correct the mutations using the BAC recombination approach if a consensus clone is needed. To demonstrate the utility of the Red/ET mediated recombination method we have generated a series of modified BACs derived from this CSFV full-length cDNA. These include BACs with substitution of the linear TAV-epitope present in the E2 protein and also BACs with substitution of the complete E2 protein with heterologous pestivirus sequences.", "These include BACs with substitution of the linear TAV-epitope present in the E2 protein and also BACs with substitution of the complete E2 protein with heterologous pestivirus sequences. We have also used the same approach for a range of different targeted modifications within CSFV BACs including specific deletions and substitutions in the 5′UTR of CSFV [24] and for insertions of heterologous reporter sequences into CSFV replicons [25] . Using Red/ET recombinationmediated mutagenesis for the targeted design, the work can be expedited and focused, in principal, on any sequence within the viral genome and is not dependent on the use of internal restriction sites.", "Using Red/ET recombinationmediated mutagenesis for the targeted design, the work can be expedited and focused, in principal, on any sequence within the viral genome and is not dependent on the use of internal restriction sites. The results demonstrate that Red/ ET recombination-mediated mutagenesis of pestivirus BAC cDNAs provides a useful tool for advancing the construction of modified pestiviruses. Cells infected with the parental vR26 virus were recognized by the two anti-E2 mAbs (WH211 and WH303) specific for the CSFV-E2 proteins, in contrast cells infected with the modified viruses vR26_TAV and vR26_E2gif, rescued from the recombined BACs, were not detected by these mAbs.", "Cells infected with the parental vR26 virus were recognized by the two anti-E2 mAbs (WH211 and WH303) specific for the CSFV-E2 proteins, in contrast cells infected with the modified viruses vR26_TAV and vR26_E2gif, rescued from the recombined BACs, were not detected by these mAbs. Furthermore, as expected, cells infected with the vR26_E2gif were recognized by the anti-BDV mAb (WB166) whereas no staining was observed with this antibody in vR26 infected cells or in cells with vR26_TAV. The mAb WH303 recognizes the CSFV TAV-epitope [18] and the difference in 4 aa between the TAV-epitope and the corresponding sequence from BDV strain \"Gifhorn\" is enough to completely abolish the recognition by this mAb.", "The mAb WH303 recognizes the CSFV TAV-epitope [18] and the difference in 4 aa between the TAV-epitope and the corresponding sequence from BDV strain \"Gifhorn\" is enough to completely abolish the recognition by this mAb. The lack of staining of vR26_TAV infected cells by the WH211 indicated that the TAV-sequence is also important for the epitope recognized by this mAb. Thus, the chimeric pestiviruses, vR26_TAV and vR26_E2gif, containing heterologous E2 sequences can be readily discriminated from the vR26 using specific anti-E2 monoclonal antibodies.", "Thus, the chimeric pestiviruses, vR26_TAV and vR26_E2gif, containing heterologous E2 sequences can be readily discriminated from the vR26 using specific anti-E2 monoclonal antibodies. These new chimeric pestiviruses represents Cstrain based marked vaccine candidates with the characteristics desired for safe and efficacious DIVA vaccines against CSFV. Indeed, vR26_E2gif vaccinated pigs could be efficiently discriminated from C-strain vaccinated pigs and from CSFV infected pigs using CSFV-E2 specific antibody ELISAs (Rasmussen et al., unpublished results).", "Indeed, vR26_E2gif vaccinated pigs could be efficiently discriminated from C-strain vaccinated pigs and from CSFV infected pigs using CSFV-E2 specific antibody ELISAs (Rasmussen et al., unpublished results). Nucleotide sequence data for the pBeloR26 showed a number of changes from the published reference sequence for \"C-strain Riems\". Some of these differences are present in the cDNA derived from the vaccine stock at a detectable level whereas others may represent low-level variants within the cDNA or errors introduced by the RT-PCR amplification.", "Some of these differences are present in the cDNA derived from the vaccine stock at a detectable level whereas others may represent low-level variants within the cDNA or errors introduced by the RT-PCR amplification. Full-length sequencing revealed that no changes occurred in the cDNA during extensive propagation in E. coli DH10B of the pBeloR26 and the E2chimeric derivative, pBeloR26_E2gif, indicating a very high stability of these BAC-cloned CSFV cDNAs. This is essential if this system is to be useful for cloning and sequence manipulation, and contrasts with stability problems encountered with conventional plasmids containing fulllength pestivirus cDNAs [31] .", "This is essential if this system is to be useful for cloning and sequence manipulation, and contrasts with stability problems encountered with conventional plasmids containing fulllength pestivirus cDNAs [31] . The stability of these BACs is consistent with previous reports on the stability of BACs containing other viruses of the family Flaviviridae in E. coli [8, 10] . Extensive passaging of the rescued vR26 and the chimeric virus derivative, vR26_E2gif, resulted in a change at nucleotide position A10665G (resulting in the predicted aa" ]

[ "BACKGROUND: Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria.", "This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. RESULTS: A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria.", "Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that modifications in the E2 protein coding sequence were stably maintained. A single amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence.", "A single amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence. CONCLUSIONS: These results show that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses. Text: Bacterial artificial chromosomes (BACs) are ideally suited for the stable maintenance of large DNA sequences derived from viral genomes [1] .", "Text: Bacterial artificial chromosomes (BACs) are ideally suited for the stable maintenance of large DNA sequences derived from viral genomes [1] . A considerable number of BAC systems have been established for large DNA viruses; in particular many different herpesvirus genomes have been cloned into BACs (for review see [2] ). The first BAC systems using RNA virus cDNAs were described for coronaviruses [3] [4] [5] [6] and recently the first BAC containing a full-length cDNA for a negative-stranded RNA virus was described [7] .", "The first BAC systems using RNA virus cDNAs were described for coronaviruses [3] [4] [5] [6] and recently the first BAC containing a full-length cDNA for a negative-stranded RNA virus was described [7] . Similarly, cDNAs corresponding to the full-length genomes of members of the Flaviviridae family (Japanese encephalitis virus [8] and Dengue virus [9] ) have been inserted into BACs. BACs containing full-length cDNAs of pestiviruses (also within the Flaviviridae), including bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) have recently been established [10, 11] .", "BACs containing full-length cDNAs of pestiviruses (also within the Flaviviridae), including bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) have recently been established [10, 11] . Infectious pestiviruses can be rescued using RNA transcripts derived from these BACs. The pestiviruses have single stranded positive sense RNA genomes, about 12.3 kb in length, which includes a single long open reading frame, encoding a large polyprotein, flanked by 5′ and 3′ untranslated regions (UTRs) that are critical for autonomous replication of the genome [12, 13] .", "The pestiviruses have single stranded positive sense RNA genomes, about 12.3 kb in length, which includes a single long open reading frame, encoding a large polyprotein, flanked by 5′ and 3′ untranslated regions (UTRs) that are critical for autonomous replication of the genome [12, 13] . The polyprotein is cleaved by cellular and viral proteases into four structural proteins (nucleocapsid protein C, envelope glycoproteins E rns , E1 and E2) and eight nonstructural proteins (N pro , p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). The availability of genetically defined and stable pestivirus BACs facilitates the functional study of viral proteins or RNA structures and also the development of new marker vaccine candidates.", "The availability of genetically defined and stable pestivirus BACs facilitates the functional study of viral proteins or RNA structures and also the development of new marker vaccine candidates. Several CSFV vaccines with marker properties based on chimeric pestiviruses have been developed over the years [14] . In particular, chimeric pestiviruses with substitution of the entire E2 protein have been described [15] [16] [17] but also mutants with more subtle modifications, such as the modification of the important TAV-epitope [18] within the CSFV-E2 protein [19, 20] are promising marker vaccine candidates.", "In particular, chimeric pestiviruses with substitution of the entire E2 protein have been described [15] [16] [17] but also mutants with more subtle modifications, such as the modification of the important TAV-epitope [18] within the CSFV-E2 protein [19, 20] are promising marker vaccine candidates. Manipulation of BACs using traditional cloning procedures can be difficult (e.g. because of a lack of convenient restriction enzyme sites) and thus a range of methodologies that apply bacterial genetics, including homologous recombination (e.g.", "because of a lack of convenient restriction enzyme sites) and thus a range of methodologies that apply bacterial genetics, including homologous recombination (e.g. Red/ET homologous recombineering) within the E. coli host, have been developed (for review, see [21] ). The use of homologous recombination allows site-directed mutagenesis of BACs [22] and, by employing a counterselection scheme, specific modifications can be obtained without leaving residual \"foreign\" sequences [23] .", "The use of homologous recombination allows site-directed mutagenesis of BACs [22] and, by employing a counterselection scheme, specific modifications can be obtained without leaving residual \"foreign\" sequences [23] . The main advantage of this method is that there are no target limitations (e.g. based on size or location) and no need for suitable restriction sites.", "based on size or location) and no need for suitable restriction sites. The integration of the modified sequence is performed in vivo (within E. coli) thereby potentially being more accurate than in vitro approaches like PCR-based methods. Although in vitro cloning approaches based on the use of high-fidelity polymerases for PCR amplification have significantly improved in recent years, the use of in vivo approaches should allow a more accurate method of mutagenesis due to the use of the cells own high-fidelity replication system which includes proof reading.", "Although in vitro cloning approaches based on the use of high-fidelity polymerases for PCR amplification have significantly improved in recent years, the use of in vivo approaches should allow a more accurate method of mutagenesis due to the use of the cells own high-fidelity replication system which includes proof reading. Whereas BAC recombination has been commonly used for modifying DNA viruses, there are only very few reports about the use of this technology for RNA viruses [7, 24, 25] . Here, a generally applicable strategy for the manipulation and rescue of chimeric pestiviruses from BACs is described as a model, and the flexibility of this approach is demonstrated by generating different modifications in the viral cDNA of the new CSFV-BAC, pBeloR26, derived from the modified live vaccine strain \"C-strain Riems\".", "Here, a generally applicable strategy for the manipulation and rescue of chimeric pestiviruses from BACs is described as a model, and the flexibility of this approach is demonstrated by generating different modifications in the viral cDNA of the new CSFV-BAC, pBeloR26, derived from the modified live vaccine strain \"C-strain Riems\". The targeted recombination-mediated mutagenesis described here includes the substitution of the 9 amino acid (aa) linear TAV-epitope (TAVSPTTLR) present in the E2 protein with the corresponding region (TTVSTSTLA) of a heterologous pestivirus (border disease virus, BDV, strain \"Gifhorn\") and also the replacement of the entire CSFV E2 protein coding region with the whole E2 coding region from the same BDV, to generate marked vaccine viruses that can be discriminated using specific anti-E2 monoclonal antibodies. The genetic stabilities of both the BAC constructs (within E. coli) and the rescued viruses have also been assessed.", "The genetic stabilities of both the BAC constructs (within E. coli) and the rescued viruses have also been assessed. Porcine kidney (PK15) and sheep fetal thymoid (SFT-R) cells were grown at 37°C (with 5% (v/v) CO 2 ) in Dulbecco's minimal essential medium (DMEM) supplemented with 5% (v/v) pestivirus-free fetal calf serum. Virus from a bait containing the modified live vaccine CSFV \"C-strain Riems\" (Riemser Arzneimittel AG, Germany) was propagated once in PK15 cells and termed vRiemser.", "Virus from a bait containing the modified live vaccine CSFV \"C-strain Riems\" (Riemser Arzneimittel AG, Germany) was propagated once in PK15 cells and termed vRiemser. RNA obtained from BDV strain \"Gifhorn\" [26] was used for amplification of the Gifhorn E2-coding sequence. Oligonucleotide primers used are listed in Additional file 1: Table S1 .", "Oligonucleotide primers used are listed in Additional file 1: Table S1 . The BAC construct, pBeloR26, was constructed using the long RT-PCR method as previously described [11] using RNA derived from the \"C-strain Riems\". Briefly, full-length viral cDNAs flanked by NotI sites were amplified by long RT-PCR using primers 5′Cstrain_T7_Not1 (which includes a T7 promotor for in vitro transcription, a NotI site and a region corresponding to the first 44 nt of the genome) and 3′CSFV_Not1 (that contains a NotI site and sequence complementary to the 3′-terminal 35 nt of the genome that are conserved among many CSFVs including the Cstrain).", "Briefly, full-length viral cDNAs flanked by NotI sites were amplified by long RT-PCR using primers 5′Cstrain_T7_Not1 (which includes a T7 promotor for in vitro transcription, a NotI site and a region corresponding to the first 44 nt of the genome) and 3′CSFV_Not1 (that contains a NotI site and sequence complementary to the 3′-terminal 35 nt of the genome that are conserved among many CSFVs including the Cstrain). The product (ca. 12.3 kbp) was digested with NotI and inserted into similarly digested pBeloBAC11 (New England Biolabs, GenBank accession U51113).", "12.3 kbp) was digested with NotI and inserted into similarly digested pBeloBAC11 (New England Biolabs, GenBank accession U51113). All BACs were modified and maintained in E. coli DH10B cells (Invitrogen) grown at 37°C in LB medium containing chloramphenicol (Cam, 15 μg/ml). The electroporation of bacteria was performed in 0.1 cm cuvettes using 1 pulse at 1800 V, 25 μF and 200 Ω in a Gene Pulser Xcell (Bio-Rad).", "The electroporation of bacteria was performed in 0.1 cm cuvettes using 1 pulse at 1800 V, 25 μF and 200 Ω in a Gene Pulser Xcell (Bio-Rad). BACs to be used as templates for long PCR or for screening by restriction enzyme digestion were purified from 4 ml overnight cultures of E. coli DH10B using the ZR BAC DNA Miniprep Kit (Zymo Research). BACs required for direct genome sequencing were purified from 500 ml cultures using the Large-construct kit (Qiagen).", "BACs required for direct genome sequencing were purified from 500 ml cultures using the Large-construct kit (Qiagen). Modifications to the full-length CSFV cDNA were accomplished in E. coli DH10B (streptomycin resistant, Strep R ) using the Counter Selection BAC Modification Kit (Gene Bridges, Heidelberg, Germany). The Red/ET recombination involved three steps (i-iii).", "The Red/ET recombination involved three steps (i-iii). Step i) the temperature-sensitive pRedET expression plasmid (Gene Bridges) was introduced into electroporationcompetent E.coli DH10B cells containing the parental BAC (phenotype Cam R , Strep R ). The pRedET expresses the phage lambda proteins redα, redβ and redγ, under control of the arabinose-inducible pBAD promoter, allowing homologous recombination to occur.", "The pRedET expresses the phage lambda proteins redα, redβ and redγ, under control of the arabinose-inducible pBAD promoter, allowing homologous recombination to occur. Immediately after electroporation, pre-warmed LB medium without antibiotics (1 ml) was added to the cells which were then incubated at 30°C for 1 hour, prior to spreading onto agar plates containing Cam (15 μg/ml) and tetracycline (Tet) (3 μg/ml) and then incubated at 30°C overnight to maintain the pRedET. The presence of the pRedET plasmid (conferring Tet R ) was verified by visual inspection of BAC-DNA preparations from the Cam R /Tet R colonies using agarose gel electrophoresis.", "The presence of the pRedET plasmid (conferring Tet R ) was verified by visual inspection of BAC-DNA preparations from the Cam R /Tet R colonies using agarose gel electrophoresis. Step ii) counter-selection marker cassettes with an extra NotI site for screening purposes (rpsL-neo, 1325 bp) were amplified by PCR using primers with 30 nt or 50 nt extensions that were homologous to the target site in the BAC using the rpsL-neo plasmid (Gene Bridges) as template and the Phusion hot start II HF DNA polymerase (Thermo Scientific) with cycling conditions as follows: 98°C for 30s, followed by 35 cycles of 98°C for 10s, 60°C for 20s, 72°C for 60s, and 1 cycle at 72°C for 4 min. The PCR products (ca.", "The PCR products (ca. 1400 bp) were isolated on 1% (w/v) TBE agarose gels and purified using a GeneJET gel extraction kit (Thermo Scientific). Samples (30 μl), from an E. coli culture containing pRedET and the parental BAC grown overnight at 30°C in LB media (Cam, Tet), were used to inoculate 1.4 ml of fresh LB media with the same antibiotics to obtain exponentially growing bacteria at 30°C.", "Samples (30 μl), from an E. coli culture containing pRedET and the parental BAC grown overnight at 30°C in LB media (Cam, Tet), were used to inoculate 1.4 ml of fresh LB media with the same antibiotics to obtain exponentially growing bacteria at 30°C. Red/ET recombination proteins were induced by adding 50 μl of 10% (w/v) L-arabinose (Sigma). The PCR product (200 ng) containing the rpsL-neo cassette was introduced into these bacteria using electroporation (as above).", "The PCR product (200 ng) containing the rpsL-neo cassette was introduced into these bacteria using electroporation (as above). Following electroporation, the cells were grown at 37°C for 70 min (to allow recombination) and then selected on plates containing Cam (15 μg/ml), Tet (3 μg/ml) and kanamycin (Kan, 15 μg/ml) overnight at 30°C to maintain the pRedET. Note, the rpsL cassette confers Streptomycin sensitivity (Strep S ) onto the resistant DH10B strain and the neo confers Kanamycin resistance (Kan R ).", "Note, the rpsL cassette confers Streptomycin sensitivity (Strep S ) onto the resistant DH10B strain and the neo confers Kanamycin resistance (Kan R ). The correct phenotype (Cam R , Kan R , Tet R , Strep S ) of the resulting colonies was confirmed by streaking the colonies onto plates containing Cam (15 μg/ml), Tet (3 μg/ml) and Kan (15 μg/ml) and grown at 30°C. Importantly, for the third step, the replacement of the rpsL-neo cassette (using counter-selection), the selected colonies were also streaked onto plates containing Cam (15 μg/ml) plus Strep (50 μg/ml) and shown to be Strep S indicating incorporation of a functional rpsL gene.", "Importantly, for the third step, the replacement of the rpsL-neo cassette (using counter-selection), the selected colonies were also streaked onto plates containing Cam (15 μg/ml) plus Strep (50 μg/ml) and shown to be Strep S indicating incorporation of a functional rpsL gene. The structures of the intermediate BACs were verified by restriction enzyme analysis and sequencing around the inserts. Step iii) the replacement of the rpsL-neo selection cassettes from the intermediate constructs using linear DNA fragments was achieved through counter-selection and Red/ET recombination.", "Step iii) the replacement of the rpsL-neo selection cassettes from the intermediate constructs using linear DNA fragments was achieved through counter-selection and Red/ET recombination. Again, the homologous sequences at the ends of the DNA fragment were used for Red/ET mediated recombination events to replace the rpsL-neo cassette with the sequence of interest. Counterselection against the rpsL-neo cassette (phenotype Cam R , Kan R , Tet R , Strep S ) was employed using media containing Cam (15 μg/ml) and Strep (50 μg/ml) to isolate the required derivatives (phenotype Cam R and Strep R ).", "Counterselection against the rpsL-neo cassette (phenotype Cam R , Kan R , Tet R , Strep S ) was employed using media containing Cam (15 μg/ml) and Strep (50 μg/ml) to isolate the required derivatives (phenotype Cam R and Strep R ). Initially, the intermediate construct, pBeloR26_E2rpsLneo ( Figure 1 ), was generated using Red/ET recombination by insertion of the rpsL-neo cassette with an extra NotI site for screening purposes which was amplified using primers Criems-TAVfor and Criems-TAVrev (Additional file 1: Table S1 ) in place of the TAVSPTTLR coding sequence (27 nt) . Secondly, the rpsL-neo cassette in this intermediate construct was then replaced using counter-selection Red/ ET recombination using a single-stranded oligonucleotide, Riems_TAV_Gifhorn (Additional file 1: Table S1 ) with the same homology arms as used for the rpsL-neo cassette, to introduce the coding sequence for the BDV \"Gifhorn\" epitope sequence (TTVSTSTLA).", "Secondly, the rpsL-neo cassette in this intermediate construct was then replaced using counter-selection Red/ ET recombination using a single-stranded oligonucleotide, Riems_TAV_Gifhorn (Additional file 1: Table S1 ) with the same homology arms as used for the rpsL-neo cassette, to introduce the coding sequence for the BDV \"Gifhorn\" epitope sequence (TTVSTSTLA). The resulting construct was named pBeloR26_TAV (Figure 1 ). The initial intermediate construct (with rpsL-neo) was then used to produce the pBeloR26_E2gif construct ( Figure 1 ).", "The initial intermediate construct (with rpsL-neo) was then used to produce the pBeloR26_E2gif construct ( Figure 1 ). For this, the E2 coding sequence was amplified from cDNA prepared from BDV \"Gifhorn\" RNA using two different primer pairs, one set with 50 nt homology arms (Criems_E2_gifFlong/Criems_ E2_gifRlong) and another with 30 nt homologous sequences (Criems_E2_gifF/Criems_E2_gifR). For generation of BACs with substitution of the entire E2 coding sequences, PCR products consisting of the sequence of interest flanked with homology arms identical to the target area were generated by PCR (as for the rpsLneo cassette).", "For generation of BACs with substitution of the entire E2 coding sequences, PCR products consisting of the sequence of interest flanked with homology arms identical to the target area were generated by PCR (as for the rpsLneo cassette). For making constructs with substitution of shorter sequences (e.g. the TAV-epitope), the recombination was achieved using synthetic single stranded oligonucleotides rather than PCR products.", "the TAV-epitope), the recombination was achieved using synthetic single stranded oligonucleotides rather than PCR products. Pre-heating of single stranded oligonucleotides at 95°C for 2 min followed by snap-freezing, prior to electroporation, empirically showed the best results. In each case, the DNA molecules were introduced into E. coli containing the BAC derivatives including the rpsL-neo cassettes together with the pRedET plasmid by electroporation as described above.", "In each case, the DNA molecules were introduced into E. coli containing the BAC derivatives including the rpsL-neo cassettes together with the pRedET plasmid by electroporation as described above. The structures of the modified BACs were verified by restriction enzyme analysis and subsequent full-genome sequencing (see below). BAC DNA (1 μg) was linearized with NotI or 1 μl BAC DNA was used as template for long PCR amplification using primers 5′C-strain_T7_Not1 and 3′CSFV (Additional file 1: Table S1 ).", "BAC DNA (1 μg) was linearized with NotI or 1 μl BAC DNA was used as template for long PCR amplification using primers 5′C-strain_T7_Not1 and 3′CSFV (Additional file 1: Table S1 ). Linearized BACs or PCR products were purified with the GeneJet PCR purification kit (Thermo Scientific) and transcribed in vitro using a Megascript T7 kit (Invitrogen). Viruses were rescued from RNA transcripts (1 to 5 μg) by electroporation of porcine (PK15) or ovine (SFT-R) cells essentially as described previously [24] .", "Viruses were rescued from RNA transcripts (1 to 5 μg) by electroporation of porcine (PK15) or ovine (SFT-R) cells essentially as described previously [24] . Cells were analysed using immunofluorescence microscopy (typically after 3 days) for the expression of NS3 and E2 proteins using specific monoclonal antibodies (mAbs), these were anti-NS3 (WB103/105, pan-pestivirus), anti-CSFV E2 (WH211, WH303, both CSFV specific) and anti-BDV E2 (WB166, BVDV/BDV specific) (AHVLA Scientific, United Kingdom) together with Alexa 488 conjugated goat antimouse IgG antibody (Molecular Probes, Invitrogen). The nuclei of cells were visualized using DAPI (Vector Laboratories) and images were recorded using a BX63 fluorescence microscope (Olympus).", "The nuclei of cells were visualized using DAPI (Vector Laboratories) and images were recorded using a BX63 fluorescence microscope (Olympus). For peroxidase staining, cells were fixed and stained for the presence of pestivirus antigens using biotinylated pig anti-CSFV/BVDV polyclonal IgG followed by avidin-conjugated horseradish peroxidase (eBioscience) as previously described [27] . The same staining procedure was also performed using the anti-E2 mAbs.", "The same staining procedure was also performed using the anti-E2 mAbs. Samples containing virus-positive cells were passaged onto new cells. Virus growth curves were generated as previously described [24] . Briefly, PK15 or SFT-R cells were infected at a multiplicity of infection (MOI) of 0.1 pfu/cell and grown for three days.", "Briefly, PK15 or SFT-R cells were infected at a multiplicity of infection (MOI) of 0.1 pfu/cell and grown for three days. BAC DNAs (5 μg), purified using the Large-construct kit (Qiagen), or PCR products (1 μg) amplified from viral cDNA or from BACs using the long PCR method (as above) were consensus sequenced using a 454 FLX (Roche) or an Ion PGM (Life Technologies). Both Newbler (Roche) and the bwa.bwasw alignment algorithm [28] were used for mapping the reads to the expected sequence.", "Both Newbler (Roche) and the bwa.bwasw alignment algorithm [28] were used for mapping the reads to the expected sequence. A combination of Samtools [29] and LoFreq SNV-caller [30] was used for downstream single nucleotide variant (SNV) analysis. Finally, clone consensus sequences were aligned using MAFFT in the Geneious software platform (Biomatters).", "Finally, clone consensus sequences were aligned using MAFFT in the Geneious software platform (Biomatters). Generation of a BAC containing full-length cDNA corresponding to the modified live vaccine \"C-strain Riems\"\n\nBACs containing the full-length cDNA corresponding to the parental vRiemser (\"C-strain Riems\") were constructed according to the method described previously for the \"Paderborn\" strain of CSFV [11] . BACs containing the complete CSFV cDNAs were identified by restriction Figure 1 Schematic representation of the CSFV genome organization and the BACs constructed and used in this study.", "BACs containing the complete CSFV cDNAs were identified by restriction Figure 1 Schematic representation of the CSFV genome organization and the BACs constructed and used in this study. Nucleotide (nt) and amino acid (aa) positions within R26 for the 5′ and 3′ termini together with the translational start and stop codons of the polyprotein coding region plus cleavage sites used to make the individual proteins (N pro , C, E rns , E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) are indicated. Insertion of the rpsL-neo in place of the TAV-epitope within CSFV E2 for the intermediate construct (R26_rpsLneo) and the subsequent replacement with the TTVSTSTLA sequence (R26_TAV) and the complete substitution of the E2 sequence (R26_E2gif) are shown.", "Insertion of the rpsL-neo in place of the TAV-epitope within CSFV E2 for the intermediate construct (R26_rpsLneo) and the subsequent replacement with the TTVSTSTLA sequence (R26_TAV) and the complete substitution of the E2 sequence (R26_E2gif) are shown. Names of BAC constructs begin with \"pBelo\" and rescued viruses with \"v\" (e.g. pBeloR26 and vR26).", "pBeloR26 and vR26). Cell culture passage no. of virus is indicated with \"/P\" (e.g. vR26/P-4). digest analysis and following linearization by NotI, RNA transcripts were produced and electroporated into PK15 cells.", "digest analysis and following linearization by NotI, RNA transcripts were produced and electroporated into PK15 cells. This screening resulted in the identification of a BAC containing a cDNA insert of 12316 nt, pBeloR26 (Figure 1) , which yielded infectious virus, termed vR26, that could be propagated in SFT-R cells (Figure 2 , upper panels) and in PK15 cells (Figure 3 ). The rescued vR26 displayed higher growth rate at the early stage (about 10fold difference in virus yield at 24 h) compared to the parental vaccine virus, but after 48 hours similar virus titres were obtained (Figure 3 ).", "The rescued vR26 displayed higher growth rate at the early stage (about 10fold difference in virus yield at 24 h) compared to the parental vaccine virus, but after 48 hours similar virus titres were obtained (Figure 3 ). Full-genome sequencing of the cloned BAC template, pBeloR26, revealed a number of differences throughout the genome when compared to the full-length consensus sequence of the cDNA used for the cloning procedure (see Table 1 ). These differences are non-representative variants within the cDNA.", "These differences are non-representative variants within the cDNA. Overall, the BAC sequence differed from the cDNA sequence in 18 positions, 9 of these lead to predicted amino acid substitutions within the polyprotein; one in each of N pro , E rns , E1, E2 and NS3 and four amino acid substitutions in NS5B (Table 1) . When compared to the published reference sequence (GenBank accession AY259122.1), the pBeloR26 BAC sequence differed at an additional 11 positions, 1 of these lead to a predicted amino acid substitution and there was one large insertion (27 nt) in the hypervariable region of the 3′-UTR (Additional file 2: Table S2 ).", "When compared to the published reference sequence (GenBank accession AY259122.1), the pBeloR26 BAC sequence differed at an additional 11 positions, 1 of these lead to a predicted amino acid substitution and there was one large insertion (27 nt) in the hypervariable region of the 3′-UTR (Additional file 2: Table S2 ). To determine the utility of the targeted recombinationmediated mutagenesis system for pestiviruses, two different modifications of the E2 protein coding sequence within pBeloR26 were generated using the Red/ET recombination methodology. Initially, the sequence encoding the linear TAV-epitope (TAVSPTTLR) within the CSFV-E2 was substituted with the sequence encoding the corresponding region (encoding TTVSTSTLA) from the BDV strain \"Gifhorn\" as described in the Materials and Methods section.", "Initially, the sequence encoding the linear TAV-epitope (TAVSPTTLR) within the CSFV-E2 was substituted with the sequence encoding the corresponding region (encoding TTVSTSTLA) from the BDV strain \"Gifhorn\" as described in the Materials and Methods section. More than 90% of the colonies obtained using this procedure contained the required BAC\n\nAnti-CSFV E2 (WH211) Figure 2 Antibody reaction patterns of pestivirus infected cells. SFT-R cells were infected with vR26 and its two derivatives vR26_E2gif and vR26_TAV plus vGifhorn [26] .", "SFT-R cells were infected with vR26 and its two derivatives vR26_E2gif and vR26_TAV plus vGifhorn [26] . After 72 h, the cells were fixed and stained with monoclonal antibodies against the NS3 protein (WB103/105, left column), the CSFV E2 protein (WH303 and WH211, middle columns) and the BDV E2 protein (WB166, right column) as indicated and viewed using a fluorescence microscope. structure as determined by NotI digestions.", "structure as determined by NotI digestions. The complete genome sequences of the CSFV cDNA within two selected BACs, designated pBeloR26_TAV have been verified (data not shown). In addition, the complete coding sequence (1119 nt) for the CSFV-E2 protein was substituted by the corresponding sequence from BDV \"Gifhorn\".", "In addition, the complete coding sequence (1119 nt) for the CSFV-E2 protein was substituted by the corresponding sequence from BDV \"Gifhorn\". Again more than 90% of the colonies obtained contained the required BAC and the same proportion of correctly recombined BACs was obtained using either 30 nt or 50 nt homology arms. The chimeric BAC was designated, pBeloR26_E2gif and the complete virus genome sequence (cDNA) was verified (data not shown).", "The chimeric BAC was designated, pBeloR26_E2gif and the complete virus genome sequence (cDNA) was verified (data not shown). After electroporation with RNA transcripts derived from either pBeloR26_TAV or pBeloR26_E2gif a large number of CSFV NS3-positive cells could be observed (data not shown) and chimeric virus stocks, termed vR26_TAV and vR26_E2gif, were generated after further passages in cells. Cells infected with these viruses and with the parental vR26 and vGifhorn strains were all stained with mAbs directed against the NS3 protein ( Figure 2 ).", "Cells infected with these viruses and with the parental vR26 and vGifhorn strains were all stained with mAbs directed against the NS3 protein ( Figure 2 ). However, in contrast to the parental vR26 virus, the chimeric viruses rescued from the recombined BACs were not recognized by anti-E2 mAbs specific for the CSFV-E2 proteins ( Figure 2 ) and thus, consistent with their structure, displayed the same antibody reaction pattern as vGifhorn. Two different anti-CSFV E2 mAbs, WH211 and WH303, were used for the staining and the latter has been shown previously to target the TAV-epitope [18] .", "Two different anti-CSFV E2 mAbs, WH211 and WH303, were used for the staining and the latter has been shown previously to target the TAV-epitope [18] . As anticipated, cells infected with either the vGifhorn or with the chimeric vR26_E2gif could be shown to express the \"Gifhorn\" E2 protein using staining with an anti-BDV mAb ( Figure 2 ). The presence of the BDV epitope TTVSTSTLA in vR26_ TAV was insufficient to permit efficient recognition by this anti-BDV mab, although a weak signal was observed in some cells.", "The presence of the BDV epitope TTVSTSTLA in vR26_ TAV was insufficient to permit efficient recognition by this anti-BDV mab, although a weak signal was observed in some cells. The BAC constructs pBeloR26 and pBeloR26_E2gif were analysed for the genetic stability of the cDNA to determine the suitability of the BAC vector for maintaining full-length pestivirus cDNAs. E. coli DH10B cells containing the BACs were passaged 15 times, by overnight growth, and the complete viral cDNAs within the BACs were sequenced after the 1st and the 15th passage.", "E. coli DH10B cells containing the BACs were passaged 15 times, by overnight growth, and the complete viral cDNAs within the BACs were sequenced after the 1st and the 15th passage. No mutations were observed within the 12316 nt virus cDNA sequences after this extensive propagation of the BACs in the bacterial host, indicating a highly stable system for the maintenance of complete pestivirus cDNA sequences. The viruses, vR26 and vR26_E2gif, rescued from their respective BAC constructs, were also tested for their genetic stability within mammalian cells.", "The viruses, vR26 and vR26_E2gif, rescued from their respective BAC constructs, were also tested for their genetic stability within mammalian cells. Linearized BAC DNA was transcribed in vitro and the RNA was electroporated into PK15 cells. Three days after electroporation the cells were stained with the anti-NS3 antibody to detect the presence of replicating virus.", "Three days after electroporation the cells were stained with the anti-NS3 antibody to detect the presence of replicating virus. Samples containing virus positive cells were passaged onto new cells, this process \n\n*Nt position 10665 in vR26/P-12 is reverted from A to G as in the parental cDNA. was repeated for 12 separate passages (each of three days).", "was repeated for 12 separate passages (each of three days). The virus titre (as TCID 50 /ml) was determined for each passage. Passage of the rescued vR26_E2gif chimeric virus in PK15 cells resulted in rapidly decreasing virus titres and was discontinued after the 2nd passage ( Figure 4A ).", "Passage of the rescued vR26_E2gif chimeric virus in PK15 cells resulted in rapidly decreasing virus titres and was discontinued after the 2nd passage ( Figure 4A ). Instead, further passage of this chimeric virus was performed in ovine SFT-R cells (the preferred cell type for BDV) and resulted in much higher titers of the chimeric virus. Virus titers reached more than 10 6 TCID 50 /ml after the 1st passage and remained stable for 12 passages ( Figure 4A ).", "Virus titers reached more than 10 6 TCID 50 /ml after the 1st passage and remained stable for 12 passages ( Figure 4A ). The rescued vR26 was also efficiently propagated on the SFT-R cells but maintained a slightly lower titer than the vR26_E2gif chimeric virus ( Figure 4A ). To check that the viruses retained their antibody reaction properties ( Figure 2 ) after these passages, cells were infected with viruses from the 12th SFT-R cell culture passage (termed vR26/P-12 and vR26_E2gif/P-12) and stained with a polyclonal anti-pestivirus serum and with specific mAbs directed against the CSFV-E2 and BDV-E2 proteins ( Figure 4B ).", "To check that the viruses retained their antibody reaction properties ( Figure 2 ) after these passages, cells were infected with viruses from the 12th SFT-R cell culture passage (termed vR26/P-12 and vR26_E2gif/P-12) and stained with a polyclonal anti-pestivirus serum and with specific mAbs directed against the CSFV-E2 and BDV-E2 proteins ( Figure 4B ). Cells infected with either the vR26/P-12 or the chimeric vR26_E2gif/P-12 were each detected by the polyclonal anti-pestivirus serum as expected. The anti-CSFV-E2 mAb specifically detected cells infected with vR26/P-12 but not cells infected by the chimeric virus containing the BDV-E2 protein (consistent with the results shown in Figure 2 ).", "The anti-CSFV-E2 mAb specifically detected cells infected with vR26/P-12 but not cells infected by the chimeric virus containing the BDV-E2 protein (consistent with the results shown in Figure 2 ). In contrast, the anti-BDV-E2 mAb specifically detected infection by the vR26_E2gif/P-12 and did not recognize cells infected with vR26/P-12. Each result is in accord with the structure of the viruses.", "Each result is in accord with the structure of the viruses. The 4th passage of vR26 (vR26/P-4) displayed a slower growth rate than the virus obtained after 12 passages (see Figure 5A ). It also had a reduced growth rate compared to both the vR26_E2gif/P-4 and vR26_E2gif/P-12.", "It also had a reduced growth rate compared to both the vR26_E2gif/P-4 and vR26_E2gif/P-12. The fulllength sequence of pBeloR26 had revealed ten non-silent mutations compared to the reference sequence (AY25 9122.1) for this virus (Additional file 2: Table S2 ). Any of these mutations could be responsible for the impaired growth acting alone or in concert.", "Any of these mutations could be responsible for the impaired growth acting alone or in concert. For further investigation of this issue, full length cDNAs prepared from vR26/ P-4, vR26/P-12, vR26_E2gif/P-4 and vR26_E2gif/P-12 were deep-sequenced using both the 454 FLX and Ion PGM platforms for comparison and to determine the quasispecies distribution (Additional file 3: Figure S1 and Additional file 4: Figure S2 ). Sequencing data from both platforms revealed that both the vR26/P-12 and vR26_E2gif/P-12 were close to 100% changed at nt position A10665G compared to the BAC clones (resulting in the predicted amino acid substitution D3431G within the NS5B protein, the RNAdependent RNA polymerase, see Figure 5B ).", "Sequencing data from both platforms revealed that both the vR26/P-12 and vR26_E2gif/P-12 were close to 100% changed at nt position A10665G compared to the BAC clones (resulting in the predicted amino acid substitution D3431G within the NS5B protein, the RNAdependent RNA polymerase, see Figure 5B ). This adaptation is a reversion back to the consensus cDNA sequence of the parental vaccine virus, vRiemser (Additional file 2: Table S2 ). Additionally, vR26/P-4 and vR26_E2gif/P-4 already showed evidence for this reversion being present within the population.", "Additionally, vR26/P-4 and vR26_E2gif/P-4 already showed evidence for this reversion being present within the population. For vR26/P-4, the level of reversion was 57%, while for vR26_E2gif/P-4 the extent of change was 73% (see Figure 5B ). In this study, we have established the first BAC containing the full-length cDNA of a CSFV vaccine strain.", "In this study, we have established the first BAC containing the full-length cDNA of a CSFV vaccine strain. The BAC differed from the parental cDNA sequence in 18 positions leading to 9 aa substitutions ( Table 1 ). The method that has been used for the generation of pBeloR26 is based on full genome amplification of cDNA followed by direct cloning to obtain the BACs [11] .", "The method that has been used for the generation of pBeloR26 is based on full genome amplification of cDNA followed by direct cloning to obtain the BACs [11] . This approach results in cDNA clones that reflect the quasispecies composition of the parental viral RNA and thus it is not guaranteed to obtain cDNA clones corresponding to the consensus sequence of the cDNA used. However, it is possible to correct the mutations using the BAC recombination approach if a consensus clone is needed.", "However, it is possible to correct the mutations using the BAC recombination approach if a consensus clone is needed. To demonstrate the utility of the Red/ET mediated recombination method we have generated a series of modified BACs derived from this CSFV full-length cDNA. These include BACs with substitution of the linear TAV-epitope present in the E2 protein and also BACs with substitution of the complete E2 protein with heterologous pestivirus sequences.", "These include BACs with substitution of the linear TAV-epitope present in the E2 protein and also BACs with substitution of the complete E2 protein with heterologous pestivirus sequences. We have also used the same approach for a range of different targeted modifications within CSFV BACs including specific deletions and substitutions in the 5′UTR of CSFV [24] and for insertions of heterologous reporter sequences into CSFV replicons [25] . Using Red/ET recombinationmediated mutagenesis for the targeted design, the work can be expedited and focused, in principal, on any sequence within the viral genome and is not dependent on the use of internal restriction sites.", "Using Red/ET recombinationmediated mutagenesis for the targeted design, the work can be expedited and focused, in principal, on any sequence within the viral genome and is not dependent on the use of internal restriction sites. The results demonstrate that Red/ ET recombination-mediated mutagenesis of pestivirus BAC cDNAs provides a useful tool for advancing the construction of modified pestiviruses. Cells infected with the parental vR26 virus were recognized by the two anti-E2 mAbs (WH211 and WH303) specific for the CSFV-E2 proteins, in contrast cells infected with the modified viruses vR26_TAV and vR26_E2gif, rescued from the recombined BACs, were not detected by these mAbs.", "Cells infected with the parental vR26 virus were recognized by the two anti-E2 mAbs (WH211 and WH303) specific for the CSFV-E2 proteins, in contrast cells infected with the modified viruses vR26_TAV and vR26_E2gif, rescued from the recombined BACs, were not detected by these mAbs. Furthermore, as expected, cells infected with the vR26_E2gif were recognized by the anti-BDV mAb (WB166) whereas no staining was observed with this antibody in vR26 infected cells or in cells with vR26_TAV. The mAb WH303 recognizes the CSFV TAV-epitope [18] and the difference in 4 aa between the TAV-epitope and the corresponding sequence from BDV strain \"Gifhorn\" is enough to completely abolish the recognition by this mAb.", "The mAb WH303 recognizes the CSFV TAV-epitope [18] and the difference in 4 aa between the TAV-epitope and the corresponding sequence from BDV strain \"Gifhorn\" is enough to completely abolish the recognition by this mAb. The lack of staining of vR26_TAV infected cells by the WH211 indicated that the TAV-sequence is also important for the epitope recognized by this mAb. Thus, the chimeric pestiviruses, vR26_TAV and vR26_E2gif, containing heterologous E2 sequences can be readily discriminated from the vR26 using specific anti-E2 monoclonal antibodies.", "Thus, the chimeric pestiviruses, vR26_TAV and vR26_E2gif, containing heterologous E2 sequences can be readily discriminated from the vR26 using specific anti-E2 monoclonal antibodies. These new chimeric pestiviruses represents Cstrain based marked vaccine candidates with the characteristics desired for safe and efficacious DIVA vaccines against CSFV. Indeed, vR26_E2gif vaccinated pigs could be efficiently discriminated from C-strain vaccinated pigs and from CSFV infected pigs using CSFV-E2 specific antibody ELISAs (Rasmussen et al., unpublished results).", "Indeed, vR26_E2gif vaccinated pigs could be efficiently discriminated from C-strain vaccinated pigs and from CSFV infected pigs using CSFV-E2 specific antibody ELISAs (Rasmussen et al., unpublished results). Nucleotide sequence data for the pBeloR26 showed a number of changes from the published reference sequence for \"C-strain Riems\". Some of these differences are present in the cDNA derived from the vaccine stock at a detectable level whereas others may represent low-level variants within the cDNA or errors introduced by the RT-PCR amplification.", "Some of these differences are present in the cDNA derived from the vaccine stock at a detectable level whereas others may represent low-level variants within the cDNA or errors introduced by the RT-PCR amplification. Full-length sequencing revealed that no changes occurred in the cDNA during extensive propagation in E. coli DH10B of the pBeloR26 and the E2chimeric derivative, pBeloR26_E2gif, indicating a very high stability of these BAC-cloned CSFV cDNAs. This is essential if this system is to be useful for cloning and sequence manipulation, and contrasts with stability problems encountered with conventional plasmids containing fulllength pestivirus cDNAs [31] .", "This is essential if this system is to be useful for cloning and sequence manipulation, and contrasts with stability problems encountered with conventional plasmids containing fulllength pestivirus cDNAs [31] . The stability of these BACs is consistent with previous reports on the stability of BACs containing other viruses of the family Flaviviridae in E. coli [8, 10] . Extensive passaging of the rescued vR26 and the chimeric virus derivative, vR26_E2gif, resulted in a change at nucleotide position A10665G (resulting in the predicted aa" ]

[ "Since the outbreak of porcine epidemic diarrhea virus (PEDV) in May 2013, U.S. swine producers have lost almost five million baby pigs. In an attempt to understand the evolution of PEDV in the United States and possibly develop a control strategy, we compared the genome sequences of a PEDV strain isolated from an infected piglet against its in vitro adapted version. The original PEDV strain was grown in Vero cells and passed 10 times serially in a MARC145 cell line.", "The original PEDV strain was grown in Vero cells and passed 10 times serially in a MARC145 cell line. The sequence analysis of the native PEDV strain and in vitro passaged virus shows that the cell culture adaptation specifically modifies PEDV spike protein whereas the open reading frame 1a/b (ORF1a/b)-encoded polyprotein, the nucleoprotein, NS3B (ORF3), and membrane and envelope proteins remain unchanged. Text: highly contagious swine disease.", "Text: highly contagious swine disease. While older pigs have a chance of survival, 80 to 100 percent of PEDV-infected piglets die within 24 h of being infected. PEDV spreads primarily through fecal-oral contact (1, 2) .", "PEDV spreads primarily through fecal-oral contact (1, 2) . Once the virus is internalized, it destroys the lining of piglets' intestines, making them incapable of digesting and deriving nutrition from milk and feed (1) . The virus causes diarrhea, vomiting, and death from dehydration and starvation (2) .", "The virus causes diarrhea, vomiting, and death from dehydration and starvation (2) . PEDV is a member of the Coronavirinae subfamily and belongs to the Alphacoronavirus genus. Its genomic size ranges from approximately 26 to 32 kb, which is relatively large for an RNA virus.", "Its genomic size ranges from approximately 26 to 32 kb, which is relatively large for an RNA virus. Although vaccines for PEDV exist in China, Japan, and South Korea, there is no approved vaccine in the United States or Europe (3) . Furthermore, PEDV is still evolving within the U.S. swine population.", "Furthermore, PEDV is still evolving within the U.S. swine population. This report briefly describes the comparison of genome sequences of a PEDV strain isolated from small intestine samples of an infected piglet and its in vitro adapted version. The original PEDV strain was dubbed NPL-PEDV/2013, grown in Vero cells, and passed 10 times in a MARC145 cell line.", "The original PEDV strain was dubbed NPL-PEDV/2013, grown in Vero cells, and passed 10 times in a MARC145 cell line. The serial in vitro passage strain was named NPL-PEDV/2013/P10. The total viral RNA was extracted by TRIzol LS reagent and sequenced by Sanger dideoxy sequencing using a primer walking technique.", "The total viral RNA was extracted by TRIzol LS reagent and sequenced by Sanger dideoxy sequencing using a primer walking technique. The raw sequences were imported into the Geneious assembler (Biomatters, CA), assembled, annotated, and compared against each other using USA/Colorado/2013 (GenBank accession no. KF272920) as a reference sequence.", "KF272920) as a reference sequence. The whole-genome sequences of NPL-PEDV/2013 and NPL-PEDV/2013/P10 contain 28,038 and 28,025 nucleotides (nt), respectively, including the 5= and 3= untranslated regions (UTR). The NPL-PEDV/2013 genome shares 99% identity with all the U.S. isolates sequenced to date and many Chinese isolates as well.", "The NPL-PEDV/2013 genome shares 99% identity with all the U.S. isolates sequenced to date and many Chinese isolates as well. The top three BLAST hits were against U.S. isolates, USA/Colora-do/2013 (GenBank accession no. KF272920), IA1 (GenBank accession no. KF468753.1), and an isolate from Iowa, 13-019349 (GenBank accession no.", "KF468753.1), and an isolate from Iowa, 13-019349 (GenBank accession no. KF267450.1). The NPL-PEDV/2013 isolate also shares 99% identity with the Chinese outbreak isolate AH2012 (GenBank accession no. KC210145).", "KC210145). When the NPL-PEDV/2013/P10 strain was compared against NPL-PEDV/2013 , the open reading frame 1a/b (ORF1a/b) polyprotein, the nucleoprotein, NS3B, and membrane and envelope proteins were found to be 100% identical at the amino acid level. In contrast, the spike gene contains six nonsynonymous single nucleotide polymorphisms, resulting in amino acid (aa) substitutions in the following positions: 375 (F¡L), 486 (T¡P), 856 (D¡E), 1081 (A¡V), 1099 (A¡S), and 1253 (Y¡D).", "In contrast, the spike gene contains six nonsynonymous single nucleotide polymorphisms, resulting in amino acid (aa) substitutions in the following positions: 375 (F¡L), 486 (T¡P), 856 (D¡E), 1081 (A¡V), 1099 (A¡S), and 1253 (Y¡D). The S1 domain of spike protein contains 2 aa substitutions, whereas the S2 domain contains 4 aa substitutions. PEDV has been shown to use porcine aminopeptidase N (pAPN) as the major receptor for cell entry (4, 5) .", "PEDV has been shown to use porcine aminopeptidase N (pAPN) as the major receptor for cell entry (4, 5) . However, Vero and MARC145 cells lack pAPN, clearly indicating that other receptors or receptor-independent pathways may be used for entry (6) . The spike protein in its trimeric conformation interacts with the cell receptor and contains numerous neutralizing antibody binding epitopes (7) .", "The spike protein in its trimeric conformation interacts with the cell receptor and contains numerous neutralizing antibody binding epitopes (7) . Analysis of the spike by PeptideCutter ( peptide_cutter/) shows that the native spike protein of NPL-PEDV/2013 has 63 trypsin and 2 chymotrypsin cleavage sites at 100% efficiency whereas NPL-PEDV/2013/P10 has lost one trypsin cleavage site but the number of chymotrypsin sites remain unchanged. This indicates that cell culture adaptation specifically modifies the PEDV spike protein; however, the immunological implications are unknown.", "This indicates that cell culture adaptation specifically modifies the PEDV spike protein; however, the immunological implications are unknown. Nucleotide sequence accession numbers. The whole-genome sequences of the NPL-PEDV/2013 and NPL-PEDV/2013/P10 strains have been deposited at DDBJ/EMBL/GenBank under accession no. KJ778615 and KJ778616." ]

[ "Since the outbreak of porcine epidemic diarrhea virus (PEDV) in May 2013, U.S. swine producers have lost almost five million baby pigs. In an attempt to understand the evolution of PEDV in the United States and possibly develop a control strategy, we compared the genome sequences of a PEDV strain isolated from an infected piglet against its in vitro adapted version. The original PEDV strain was grown in Vero cells and passed 10 times serially in a MARC145 cell line.", "The original PEDV strain was grown in Vero cells and passed 10 times serially in a MARC145 cell line. The sequence analysis of the native PEDV strain and in vitro passaged virus shows that the cell culture adaptation specifically modifies PEDV spike protein whereas the open reading frame 1a/b (ORF1a/b)-encoded polyprotein, the nucleoprotein, NS3B (ORF3), and membrane and envelope proteins remain unchanged. Text: highly contagious swine disease.", "Text: highly contagious swine disease. While older pigs have a chance of survival, 80 to 100 percent of PEDV-infected piglets die within 24 h of being infected. PEDV spreads primarily through fecal-oral contact (1, 2) .", "PEDV spreads primarily through fecal-oral contact (1, 2) . Once the virus is internalized, it destroys the lining of piglets' intestines, making them incapable of digesting and deriving nutrition from milk and feed (1) . The virus causes diarrhea, vomiting, and death from dehydration and starvation (2) .", "The virus causes diarrhea, vomiting, and death from dehydration and starvation (2) . PEDV is a member of the Coronavirinae subfamily and belongs to the Alphacoronavirus genus. Its genomic size ranges from approximately 26 to 32 kb, which is relatively large for an RNA virus.", "Its genomic size ranges from approximately 26 to 32 kb, which is relatively large for an RNA virus. Although vaccines for PEDV exist in China, Japan, and South Korea, there is no approved vaccine in the United States or Europe (3) . Furthermore, PEDV is still evolving within the U.S. swine population.", "Furthermore, PEDV is still evolving within the U.S. swine population. This report briefly describes the comparison of genome sequences of a PEDV strain isolated from small intestine samples of an infected piglet and its in vitro adapted version. The original PEDV strain was dubbed NPL-PEDV/2013, grown in Vero cells, and passed 10 times in a MARC145 cell line.", "The original PEDV strain was dubbed NPL-PEDV/2013, grown in Vero cells, and passed 10 times in a MARC145 cell line. The serial in vitro passage strain was named NPL-PEDV/2013/P10. The total viral RNA was extracted by TRIzol LS reagent and sequenced by Sanger dideoxy sequencing using a primer walking technique.", "The total viral RNA was extracted by TRIzol LS reagent and sequenced by Sanger dideoxy sequencing using a primer walking technique. The raw sequences were imported into the Geneious assembler (Biomatters, CA), assembled, annotated, and compared against each other using USA/Colorado/2013 (GenBank accession no. KF272920) as a reference sequence.", "KF272920) as a reference sequence. The whole-genome sequences of NPL-PEDV/2013 and NPL-PEDV/2013/P10 contain 28,038 and 28,025 nucleotides (nt), respectively, including the 5= and 3= untranslated regions (UTR). The NPL-PEDV/2013 genome shares 99% identity with all the U.S. isolates sequenced to date and many Chinese isolates as well.", "The NPL-PEDV/2013 genome shares 99% identity with all the U.S. isolates sequenced to date and many Chinese isolates as well. The top three BLAST hits were against U.S. isolates, USA/Colora-do/2013 (GenBank accession no. KF272920), IA1 (GenBank accession no. KF468753.1), and an isolate from Iowa, 13-019349 (GenBank accession no.", "KF468753.1), and an isolate from Iowa, 13-019349 (GenBank accession no. KF267450.1). The NPL-PEDV/2013 isolate also shares 99% identity with the Chinese outbreak isolate AH2012 (GenBank accession no. KC210145).", "KC210145). When the NPL-PEDV/2013/P10 strain was compared against NPL-PEDV/2013 , the open reading frame 1a/b (ORF1a/b) polyprotein, the nucleoprotein, NS3B, and membrane and envelope proteins were found to be 100% identical at the amino acid level. In contrast, the spike gene contains six nonsynonymous single nucleotide polymorphisms, resulting in amino acid (aa) substitutions in the following positions: 375 (F¡L), 486 (T¡P), 856 (D¡E), 1081 (A¡V), 1099 (A¡S), and 1253 (Y¡D).", "In contrast, the spike gene contains six nonsynonymous single nucleotide polymorphisms, resulting in amino acid (aa) substitutions in the following positions: 375 (F¡L), 486 (T¡P), 856 (D¡E), 1081 (A¡V), 1099 (A¡S), and 1253 (Y¡D). The S1 domain of spike protein contains 2 aa substitutions, whereas the S2 domain contains 4 aa substitutions. PEDV has been shown to use porcine aminopeptidase N (pAPN) as the major receptor for cell entry (4, 5) .", "PEDV has been shown to use porcine aminopeptidase N (pAPN) as the major receptor for cell entry (4, 5) . However, Vero and MARC145 cells lack pAPN, clearly indicating that other receptors or receptor-independent pathways may be used for entry (6) . The spike protein in its trimeric conformation interacts with the cell receptor and contains numerous neutralizing antibody binding epitopes (7) .", "The spike protein in its trimeric conformation interacts with the cell receptor and contains numerous neutralizing antibody binding epitopes (7) . Analysis of the spike by PeptideCutter ( peptide_cutter/) shows that the native spike protein of NPL-PEDV/2013 has 63 trypsin and 2 chymotrypsin cleavage sites at 100% efficiency whereas NPL-PEDV/2013/P10 has lost one trypsin cleavage site but the number of chymotrypsin sites remain unchanged. This indicates that cell culture adaptation specifically modifies the PEDV spike protein; however, the immunological implications are unknown.", "This indicates that cell culture adaptation specifically modifies the PEDV spike protein; however, the immunological implications are unknown. Nucleotide sequence accession numbers. The whole-genome sequences of the NPL-PEDV/2013 and NPL-PEDV/2013/P10 strains have been deposited at DDBJ/EMBL/GenBank under accession no. KJ778615 and KJ778616." ]

[ "Since the outbreak of porcine epidemic diarrhea virus (PEDV) in May 2013, U.S. swine producers have lost almost five million baby pigs. In an attempt to understand the evolution of PEDV in the United States and possibly develop a control strategy, we compared the genome sequences of a PEDV strain isolated from an infected piglet against its in vitro adapted version. The original PEDV strain was grown in Vero cells and passed 10 times serially in a MARC145 cell line.", "The original PEDV strain was grown in Vero cells and passed 10 times serially in a MARC145 cell line. The sequence analysis of the native PEDV strain and in vitro passaged virus shows that the cell culture adaptation specifically modifies PEDV spike protein whereas the open reading frame 1a/b (ORF1a/b)-encoded polyprotein, the nucleoprotein, NS3B (ORF3), and membrane and envelope proteins remain unchanged. Text: highly contagious swine disease.", "Text: highly contagious swine disease. While older pigs have a chance of survival, 80 to 100 percent of PEDV-infected piglets die within 24 h of being infected. PEDV spreads primarily through fecal-oral contact (1, 2) .", "PEDV spreads primarily through fecal-oral contact (1, 2) . Once the virus is internalized, it destroys the lining of piglets' intestines, making them incapable of digesting and deriving nutrition from milk and feed (1) . The virus causes diarrhea, vomiting, and death from dehydration and starvation (2) .", "The virus causes diarrhea, vomiting, and death from dehydration and starvation (2) . PEDV is a member of the Coronavirinae subfamily and belongs to the Alphacoronavirus genus. Its genomic size ranges from approximately 26 to 32 kb, which is relatively large for an RNA virus.", "Its genomic size ranges from approximately 26 to 32 kb, which is relatively large for an RNA virus. Although vaccines for PEDV exist in China, Japan, and South Korea, there is no approved vaccine in the United States or Europe (3) . Furthermore, PEDV is still evolving within the U.S. swine population.", "Furthermore, PEDV is still evolving within the U.S. swine population. This report briefly describes the comparison of genome sequences of a PEDV strain isolated from small intestine samples of an infected piglet and its in vitro adapted version. The original PEDV strain was dubbed NPL-PEDV/2013, grown in Vero cells, and passed 10 times in a MARC145 cell line.", "The original PEDV strain was dubbed NPL-PEDV/2013, grown in Vero cells, and passed 10 times in a MARC145 cell line. The serial in vitro passage strain was named NPL-PEDV/2013/P10. The total viral RNA was extracted by TRIzol LS reagent and sequenced by Sanger dideoxy sequencing using a primer walking technique.", "The total viral RNA was extracted by TRIzol LS reagent and sequenced by Sanger dideoxy sequencing using a primer walking technique. The raw sequences were imported into the Geneious assembler (Biomatters, CA), assembled, annotated, and compared against each other using USA/Colorado/2013 (GenBank accession no. KF272920) as a reference sequence.", "KF272920) as a reference sequence. The whole-genome sequences of NPL-PEDV/2013 and NPL-PEDV/2013/P10 contain 28,038 and 28,025 nucleotides (nt), respectively, including the 5= and 3= untranslated regions (UTR). The NPL-PEDV/2013 genome shares 99% identity with all the U.S. isolates sequenced to date and many Chinese isolates as well.", "The NPL-PEDV/2013 genome shares 99% identity with all the U.S. isolates sequenced to date and many Chinese isolates as well. The top three BLAST hits were against U.S. isolates, USA/Colora-do/2013 (GenBank accession no. KF272920), IA1 (GenBank accession no. KF468753.1), and an isolate from Iowa, 13-019349 (GenBank accession no.", "KF468753.1), and an isolate from Iowa, 13-019349 (GenBank accession no. KF267450.1). The NPL-PEDV/2013 isolate also shares 99% identity with the Chinese outbreak isolate AH2012 (GenBank accession no. KC210145).", "KC210145). When the NPL-PEDV/2013/P10 strain was compared against NPL-PEDV/2013 , the open reading frame 1a/b (ORF1a/b) polyprotein, the nucleoprotein, NS3B, and membrane and envelope proteins were found to be 100% identical at the amino acid level. In contrast, the spike gene contains six nonsynonymous single nucleotide polymorphisms, resulting in amino acid (aa) substitutions in the following positions: 375 (F¡L), 486 (T¡P), 856 (D¡E), 1081 (A¡V), 1099 (A¡S), and 1253 (Y¡D).", "In contrast, the spike gene contains six nonsynonymous single nucleotide polymorphisms, resulting in amino acid (aa) substitutions in the following positions: 375 (F¡L), 486 (T¡P), 856 (D¡E), 1081 (A¡V), 1099 (A¡S), and 1253 (Y¡D). The S1 domain of spike protein contains 2 aa substitutions, whereas the S2 domain contains 4 aa substitutions. PEDV has been shown to use porcine aminopeptidase N (pAPN) as the major receptor for cell entry (4, 5) .", "PEDV has been shown to use porcine aminopeptidase N (pAPN) as the major receptor for cell entry (4, 5) . However, Vero and MARC145 cells lack pAPN, clearly indicating that other receptors or receptor-independent pathways may be used for entry (6) . The spike protein in its trimeric conformation interacts with the cell receptor and contains numerous neutralizing antibody binding epitopes (7) .", "The spike protein in its trimeric conformation interacts with the cell receptor and contains numerous neutralizing antibody binding epitopes (7) . Analysis of the spike by PeptideCutter ( peptide_cutter/) shows that the native spike protein of NPL-PEDV/2013 has 63 trypsin and 2 chymotrypsin cleavage sites at 100% efficiency whereas NPL-PEDV/2013/P10 has lost one trypsin cleavage site but the number of chymotrypsin sites remain unchanged. This indicates that cell culture adaptation specifically modifies the PEDV spike protein; however, the immunological implications are unknown.", "This indicates that cell culture adaptation specifically modifies the PEDV spike protein; however, the immunological implications are unknown. Nucleotide sequence accession numbers. The whole-genome sequences of the NPL-PEDV/2013 and NPL-PEDV/2013/P10 strains have been deposited at DDBJ/EMBL/GenBank under accession no. KJ778615 and KJ778616." ]

[ "Glycyrrhizin is known to exert antiviral and anti-inflammatory effects. Here, the effects of an approved parenteral glycyrrhizin preparation (Stronger Neo-Minophafen C) were investigated on highly pathogenic influenza A H5N1 virus replication, H5N1-induced apoptosis, and H5N1-induced pro-inflammatory responses in lung epithelial (A549) cells. Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher).", "Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher). Glycyrrhizin also diminished monocyte migration towards supernatants of H5N1-infected A549 cells. The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen species and (in turn) reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication.", "The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen species and (in turn) reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication. Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease. Text: Highly pathogenic H5N1 influenza A viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] .", "Text: Highly pathogenic H5N1 influenza A viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] . At least for the first wave of an H5N1 pandemic, no sufficient amounts of adequate vaccines will be available [1] [2] [3] [4] [6] [7] [8] . Therefore, antiviral therapy for influenza A viruses including highly pathogenic H5N1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] .", "Therefore, antiviral therapy for influenza A viruses including highly pathogenic H5N1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] . The neuraminidase inhibitors oseltamivir and zanamivir as well as the adamantanes amantadin and rimantadin that interfere with the influenza M2 protein are licensed for the treament of influenza [1] [2] [3] [4] 6] . However, the use of both drug classes is limited by the emergence of resistant virus strains.", "However, the use of both drug classes is limited by the emergence of resistant virus strains. In seasonal influenza strains, the majority of H3N2 viruses and a great proportion of H1N1 viruses in humans are now considered to be amantadine-and rimantadine-resistant [10] [11] [12] [13] . Moreover, a drastic increase in oseltamivir-resistant H1N1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] .", "Moreover, a drastic increase in oseltamivir-resistant H1N1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] . Preliminary data from the United States predict a further rise for the 2008/2009 season, possibly resulting in more than 90% of the circulating H1N1 strains to be oseltamivir resistant [14] . H5N1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza A virus strains and treatment-resistant H5N1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] .", "H5N1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza A virus strains and treatment-resistant H5N1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] . More-over, parenteral agents for the treatment of seriously ill patients are missing. Glycyrrhizin, a triterpene saponine, is a constituent of licorice root.", "Glycyrrhizin, a triterpene saponine, is a constituent of licorice root. It has been found to interfere with replication and/or cytopathogenic effect (CPE) induction of many viruses including respiratory viruses such as respiratory syncytial virus, SARS coronavirus, HIV, and influenza viruses [22] [23] [24] [25] [26] [27] [28] . Moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] .", "Moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] . The severity of human H5N1 disease has been associated with hypercytokinaemia (''cytokine storm'') [29, 30] . Delayed antiviral plus immunomodulator treatment reduced H5N1-induced mortality in mice [31] .", "Delayed antiviral plus immunomodulator treatment reduced H5N1-induced mortality in mice [31] . Therefore, antiinflammatory and immunomodulatory effects exerted by glycyrrhizin may be beneficial for treatment of H5N1. Also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza A virus replication and virus-induced pro-inflammatory responses [32] [33] [34] .", "Also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza A virus replication and virus-induced pro-inflammatory responses [32] [33] [34] . Stronger Neo-Minophagen C (SNMC) is a glycyrrhizin preparation (available as tablets or parenteral formulation) that is approved in Japan for the treatment of chronic hepatic diseases and is marketed in Japan, China, Korea, Taiwan, Indonesia, India, and Mongolia. Here, we investigated the influence of SNMC on H5N1 replication, on H5N1-induced cytokine expression, on H5N1-induced cellular oxidative stress, and on critical H5N1-induced cellular signalling events in human pneumocytes (A549 cell line).", "Here, we investigated the influence of SNMC on H5N1 replication, on H5N1-induced cytokine expression, on H5N1-induced cellular oxidative stress, and on critical H5N1-induced cellular signalling events in human pneumocytes (A549 cell line). Glycyrrhizin (Stronger Neo Minophagen C) was obtained from Minophagen Pharmaceuticals Co., Ltd. (Tokyo, Japan). The influenza strain A/Vietnam/1203/04 (H5N1) was received from the WHO Influenza Centre (National Institute for Medical Research, London, UK).", "The influenza strain A/Vietnam/1203/04 (H5N1) was received from the WHO Influenza Centre (National Institute for Medical Research, London, UK). The H5N1 influenza strain A/Thailand/ 1(Kan-1)/04 was obtained from Prof. Pilaipan Puthavathana (Mahidol University, Bangkok, Thailand). Virus stocks were prepared by infecting Vero cells (African green monkey kidney; ATCC, Manassas, VA) and aliquots were stored at 280uC.", "Virus stocks were prepared by infecting Vero cells (African green monkey kidney; ATCC, Manassas, VA) and aliquots were stored at 280uC. Virus titres were determined as 50% tissue culture infectious dose (TCID 50 /ml) in confluent Vero cells in 96-well microtiter plates. A549 cells (human lung carcinoma; ATCC: CCL-185, obtained from LGC Standards GmbH, Wesel, Germany) were grown at 37uC in minimal essential medium (MEM) supplemented with 10% FBS, 100 IU/ml of penicillin and 100 mg/ml streptomycin.", "A549 cells (human lung carcinoma; ATCC: CCL-185, obtained from LGC Standards GmbH, Wesel, Germany) were grown at 37uC in minimal essential medium (MEM) supplemented with 10% FBS, 100 IU/ml of penicillin and 100 mg/ml streptomycin. Human monocytes were isolated from buffy coats of healthy donors, obtained from Institute of Transfusion Medicine and Immune Haematology, German Red Cross Blood Donor Center, Johann Wolfgang Goethe-University, Frankfurt am Main. After centrifugation on Ficoll (Biocoll)-Hypaque density gradient (Biochrom AG, Berlin, Germany), mononuclear cells were collected from the interface and washed with PBS.", "After centrifugation on Ficoll (Biocoll)-Hypaque density gradient (Biochrom AG, Berlin, Germany), mononuclear cells were collected from the interface and washed with PBS. Then, monocytes were isolated using magnetically labeled CD14 MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) following the manufacturer's instructions. Monocytes were cultivated in IMDM supplemented with 10% pooled human serum, 100 IU/ml of penicillin, and 100 mg/ml streptomycin.", "Monocytes were cultivated in IMDM supplemented with 10% pooled human serum, 100 IU/ml of penicillin, and 100 mg/ml streptomycin. The cellular viability was assessed on confluent cell layers with CellTiter-GloH Luminescent Cell Viability Assay (Promega GmbH, Mannheim, Germany) according to the manufacturers' protocol. Cell viability was expressed as percentage of non-treated control.", "Cell viability was expressed as percentage of non-treated control. To determine intracellular NP localisation, H5N1-infected A549 were fixed 8 hours p.i. for 15 min with ice-cold acetone/ methanol (40:60, Mallinckrodt Baker B.V., Deventer, The Netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in PBS) directed against the influenza A virus nucleoprotein (NP) (Millipore, Molsheim, France).", "for 15 min with ice-cold acetone/ methanol (40:60, Mallinckrodt Baker B.V., Deventer, The Netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in PBS) directed against the influenza A virus nucleoprotein (NP) (Millipore, Molsheim, France). An Alexa Fluor 488 goat anti-mouse IgG (H&L) (Invitrogen, Eugene, Oregon, USA) was used (1 h incubation, 1:1000 in PBS) as secondary antibody. Nuclei were stained using 49,6-diamidino-2phenylindole (DAPI) (Sigma-Aldrich Chemie GmbH, Munich, Germany).", "Nuclei were stained using 49,6-diamidino-2phenylindole (DAPI) (Sigma-Aldrich Chemie GmbH, Munich, Germany). Fluorescence was visualised using Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany). For flow cytometric analysis, the same antibodies were used. The cytopathogenic effect (CPE) reduction assay was performed as described before [34] .", "The cytopathogenic effect (CPE) reduction assay was performed as described before [34] . Confluent A549 cell monolayers grown in 96-well microtitre plates were infected with influenza A strains at the indicated multiplicities of infection (MOIs). After a one hour adsorption period, cells were washed to remove non-detached virus.", "After a one hour adsorption period, cells were washed to remove non-detached virus. The virus-induced CPE was recorded at 24 h post infection (p.i.). Unless otherwise stated, A549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period.", "Unless otherwise stated, A549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period. For time-ofaddition experiments, glycyrrhizin was added exclusively during the 1 h pre-incubation period, exclusively during the 1 h adsorption period, or after exclusively after the wash-out of input virus. Total RNA was isolated from cell cultures using TRI reagent (Sigma-Aldrich, Munich, Germany).", "Total RNA was isolated from cell cultures using TRI reagent (Sigma-Aldrich, Munich, Germany). Real time PCR for H5 was performed using described methods [35] . The following primers were used: sense 59 acg tat gac tac ccg cag tat tca g 39; antisense 59 aga cca gcy acc atg att gc 39; probe 6-FAM-tca aca gtg gcg agt tcc cta gca-TAMRA.", "The following primers were used: sense 59 acg tat gac tac ccg cag tat tca g 39; antisense 59 aga cca gcy acc atg att gc 39; probe 6-FAM-tca aca gtg gcg agt tcc cta gca-TAMRA. The fraction of cells with fractional DNA content (''sub-G1'' cell subpopulation) indicates cytotoxicity. Sub-G1 cells are considered to be dead (usually apoptotic) cells.", "Sub-G1 cells are considered to be dead (usually apoptotic) cells. Cells were fixed with 70% ethanol for two hours at 220uC. The cellular DNA was stained using propidium iodide (20 mg/ml) and analysed by flow cytometry (FacsCalibur, BD Biosciences, Heidelberg, Germany).", "The cellular DNA was stained using propidium iodide (20 mg/ml) and analysed by flow cytometry (FacsCalibur, BD Biosciences, Heidelberg, Germany). Caspase activation was measured using the Caspase-Glo 8, 9, or 3/7 Assays (Promega, Mannheim, Germany) following the manufacturer's instructions. Cell culture supernatants were collected and frozen at 280uC.", "Cell culture supernatants were collected and frozen at 280uC. Cytokines/chemokines were quantified by specific ELISA Duo Sets (R&D Systems GmbH, Wiesbaden, Germany) following the manufacturer's instructions. NFkB activity was investigated in H5N1 (MOI 0.01)-infected cells by quantification of the NFkB subunits Rel A (p65) and NFkB1 (p50) from nuclear extracts using the TransAM TM transcription factor DNA-binding ELISAs (Active Motif, Rixensart, Belgium).", "NFkB activity was investigated in H5N1 (MOI 0.01)-infected cells by quantification of the NFkB subunits Rel A (p65) and NFkB1 (p50) from nuclear extracts using the TransAM TM transcription factor DNA-binding ELISAs (Active Motif, Rixensart, Belgium). Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction. Cell culture supernatants were investigated for chemotactic activity by measurement of the activity to induce monocyte migration through membrane inserts in 24-well plates (pore size 8 mm; BD Biosciences, Heidelberg, Germany).", "Cell culture supernatants were investigated for chemotactic activity by measurement of the activity to induce monocyte migration through membrane inserts in 24-well plates (pore size 8 mm; BD Biosciences, Heidelberg, Germany). Monocytes (1610 6 in 100 ml of IMDM with 10% pooled human serum) were added into the cell culture inserts (upper chamber) and cell culture supernatants (300 ml), were added to the lower chamber of the well. After a 48 h incubation period, cells were fixed with 4% paraformaldehyde and permeabilised with PBS containing 0.3% Tritron X-100.", "After a 48 h incubation period, cells were fixed with 4% paraformaldehyde and permeabilised with PBS containing 0.3% Tritron X-100. Then, nuclei were stained with 49,6-diamidino-2phenylindole (DAPI). The upper side of the membrane was wiped with a wet swab to remove the cells, while the lower side of the membrane was rinsed with PBS.", "The upper side of the membrane was wiped with a wet swab to remove the cells, while the lower side of the membrane was rinsed with PBS. The number of cells at the lower side of each membrane was quantified by counting of cells from three randomly chosen sections (3.7 mm 2 ) using an Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany). Cells were lysed in Triton X-sample buffer and separated by SDS-PAGE.", "Cells were lysed in Triton X-sample buffer and separated by SDS-PAGE. Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction. Proteins were detected using specific antibodies against bactin (Sigma-Aldrich Chemie GmbH, Munich, Germany), JNK, phosphorylated JNK, p38, or phosphorylated p38, (all purchased from New England Biolabs GmbH, Frankfurt am Main, Germany) and were visualised by enhanced chemiluminescence using a commercially available kit (Amersham, Freiburg, Germany).", "Proteins were detected using specific antibodies against bactin (Sigma-Aldrich Chemie GmbH, Munich, Germany), JNK, phosphorylated JNK, p38, or phosphorylated p38, (all purchased from New England Biolabs GmbH, Frankfurt am Main, Germany) and were visualised by enhanced chemiluminescence using a commercially available kit (Amersham, Freiburg, Germany). Reactive oxygen species (ROS) were detected using the Image-iT LIVE Green Reactive Oxygen Species Kit (Molecular Probes, distributed by Invitrogen, Karlsruhe, Germany). Two groups were compared by t-test.", "Two groups were compared by t-test. More groups were compared by ANOVA with subsequent Student-Newman-Keuls test. The A549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type II pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g.", "The A549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type II pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g. 6,37,38]. If not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period.", "If not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period. Glycyrrhizin 200 mg/ml (the maximum tested concentration) did not affect A549 cell viability (data not shown) but clearly decreased CPE formation in A549 cells infected with the H5N1 influenza strain A/Thailand/1(Kan-1)/04 at MOIs of 0.01, 0.1 or 1 ( Figure 1A ). Similar results were obtained in A549 cells infected with strain A/Vietnam/1203/04 (H5N1) (Suppl.", "Similar results were obtained in A549 cells infected with strain A/Vietnam/1203/04 (H5N1) (Suppl. Figure 1A) . Staining of A549 cells for influenza A nucleoprotein 24 h after infection with strain H5N1 A/Thailand/1(Kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza A nucleoprotein positive cells ( Figure 1B) .", "Staining of A549 cells for influenza A nucleoprotein 24 h after infection with strain H5N1 A/Thailand/1(Kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza A nucleoprotein positive cells ( Figure 1B) . To examine the influence of glycyrrhizin on virus progeny, A549 cells were infected with the H5N1 influenza strain A/ Thailand/1(Kan-1)/04 at MOI 0.01 or MOI 1 and infectious virus titres were determined 24 h post infection ( Figure 1C ). While glycyrrhizin in concentrations up to 50 mg/ml did not affect H5N1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (MOI 0.01: 13-fold reduction, MOI 1: 10-fold reduction).", "While glycyrrhizin in concentrations up to 50 mg/ml did not affect H5N1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (MOI 0.01: 13-fold reduction, MOI 1: 10-fold reduction). Next, influence of glycyrrhizin on H5N1 replication was confirmed by the detection of viral (H5) RNA using quantitative PCR. Only glycyrrhizin concentrations $100 mg/ml significantly reduced Figure 1B) or H5N1 A/Vietnam/1203/04-infected (Suppl.", "Only glycyrrhizin concentrations $100 mg/ml significantly reduced Figure 1B) or H5N1 A/Vietnam/1203/04-infected (Suppl. Figure 1C ) A549 cells (MOI 0.01) 24 h post infection. Time-of-addition experiments revealed that maximal effects were achieved when glycyrrhizin was continuously present starting with a 1 h pre-incubation period ( Figure 1D ).", "Time-of-addition experiments revealed that maximal effects were achieved when glycyrrhizin was continuously present starting with a 1 h pre-incubation period ( Figure 1D ). Addition of glycyrrhizin post infection showed reduced antiviral effects while pre-incubation alone or glycyrrhizin addition during the adsorption period did not significantly affect H5N1 replication. For investigation of H5N1-induced cytokine expression, five pro-inflammatory genes were chosen that had been correlated to severity of influenza disease: CXCL10 (also known as interferon-cinducible protein 10, IP-10), interleukin 6 (IL6), interleukin 8, (IL8; also known as CXCL8), CCL2 (also known as monocyte chemoattractant protein 1, MCP-1), and CCL5 (also known as RANTES).", "For investigation of H5N1-induced cytokine expression, five pro-inflammatory genes were chosen that had been correlated to severity of influenza disease: CXCL10 (also known as interferon-cinducible protein 10, IP-10), interleukin 6 (IL6), interleukin 8, (IL8; also known as CXCL8), CCL2 (also known as monocyte chemoattractant protein 1, MCP-1), and CCL5 (also known as RANTES). A549 cells were infected with H5N1 A/Thailand/ 1(Kan-1)/04 or H5N1 A/Vietnam/1203/04 at MOI 0.01, 0.1, or 1. Glycyrrhizin treatment was performed with 25, 50, 100, or 200 mg/ml.", "Glycyrrhizin treatment was performed with 25, 50, 100, or 200 mg/ml. Cytokine expression was detected 24 h post infection by ELISA. Glycyrrhizin did not affect cytokine expression of noninfected cells (data not shown) but inhibited expression of all cytokines investigated in H5N1-infected cells in a dose-dependent manner (Figure 2, Figure 3A ).", "Glycyrrhizin did not affect cytokine expression of noninfected cells (data not shown) but inhibited expression of all cytokines investigated in H5N1-infected cells in a dose-dependent manner (Figure 2, Figure 3A ). Effects were more pronounced at lower MOIs. Notably, expression of all cytokines except IL8 was significantly inhibited after treatment with glycyrrhizin 50 mg/ml Figure 3A ) although these glycyrrhizin concentrations had no effect on H5N1 replication in A549 cells (Figure 1, Figure S1 ).", "Notably, expression of all cytokines except IL8 was significantly inhibited after treatment with glycyrrhizin 50 mg/ml Figure 3A ) although these glycyrrhizin concentrations had no effect on H5N1 replication in A549 cells (Figure 1, Figure S1 ). Cytokine expression by influenza A virus-infected respiratory cells causes recruitment of peripheral blood monocytes into the lungs of patients where they differentiate to macrophages which are thought to contribute to influenza A virus pathogenicity [5, 39] . In a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.1)-infected A549 cells through 8 mm filters.", "In a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.1)-infected A549 cells through 8 mm filters. Monocyte migration towards supernatants of H5N1-infected cells was strongly increased relative to migration towards supernatants of non-infected cells. Treatment of H5N1- infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( Figure 3B ).", "Treatment of H5N1- infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( Figure 3B ). Influenza viruses including H5N1 have been shown to induce caspase-dependent apoptosis in airway cells and this apoptosis has been correlated to the virus pathogenicity [40, 41] . Glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( Figure 4A-C) .", "Glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( Figure 4A-C) . Glycyrrhizin concentrations $100 mg/ml inhibited H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.01)-induced activation of the initiator caspases 8 and 9 as well as of the effector caspases 3/7 in A549 cells as determined 24 h post infection ( Figure 4A-C) . Lower glycyrrhizin concentrations did not affect H5N1-induced apoptosis.", "Lower glycyrrhizin concentrations did not affect H5N1-induced apoptosis. The detection of cells in sub-G1 phase resulted in similar findings ( Figure 4D ). Substances that inhibit H5N1-induced caspase 3 activation including caspase 3 inhibitors cause nuclear retention of RNP complexes [34, 42] .", "Substances that inhibit H5N1-induced caspase 3 activation including caspase 3 inhibitors cause nuclear retention of RNP complexes [34, 42] . In accordance, glycyrrhizin also interfered with nuclear export RNP at MOI 1 ( Figure S2 ). Similar results were obtained in MOI 0.01 H5N1 A/Thailand/1(Kan-1)/04infected cells ( Figure S3 ).", "Similar results were obtained in MOI 0.01 H5N1 A/Thailand/1(Kan-1)/04infected cells ( Figure S3 ). Influence of glycyrrhizin on H5N1-induced activation of nuclear factor kB (NFkB), p38, and on H5N1-induced cellular reactive oxygen species (ROS) formation Activation of NFkB, p38, and JNK have been associated with influenza A virus replication and virus-induced pro-inflammatory gene expression [34, [43] [44] [45] [46] [47] . While glycyrrhizin did not influence NFkB activity in non-infected A549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited NFkB activation in H5N1-infected cells ( Figure 5A ).", "While glycyrrhizin did not influence NFkB activity in non-infected A549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited NFkB activation in H5N1-infected cells ( Figure 5A ). Moreover, glycyrrhizin inhibited H5N1-induced phosphorylation of the MAPKs p38 and JNK ( Figure 5B ). In addition to their roles during influenza A virus replication and virus-induced cytokine/chemokine expression, NFkB, p38, and JNK are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] .", "In addition to their roles during influenza A virus replication and virus-induced cytokine/chemokine expression, NFkB, p38, and JNK are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] . Antioxidants had been already found to interfere with influenza A virus-induced signalling through NFkB, p38, and JNK, with influenza A virus replication, and with influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] . Since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with H5N1-induced ROS formation.", "Since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with H5N1-induced ROS formation. Indeed glycyrrhizin exerted clear antioxidative effects in H5N1 (MOI 0.01)-infected cells ( Figure 5C ) causing significant reduction of ROS formation already at a concentration of 25 mg/ml ( Figure 5D ). Here, we show that glycyrrhizin inhibits the replication of highly pathogenic H5N1 influenza A virus, H5N1-induced apoptosis, and H5N1-induced expression of pro-inflammatory cytokines in lung-derived A549 cells.", "Here, we show that glycyrrhizin inhibits the replication of highly pathogenic H5N1 influenza A virus, H5N1-induced apoptosis, and H5N1-induced expression of pro-inflammatory cytokines in lung-derived A549 cells. After intravenous administration, achievable plasma concentrations of glycyrrhizin have been described to be about 100 mg/ml [52] . Therefore, the glycyrrhizin concentrations found to interfere with H5N1 replication and H5N1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels.", "Therefore, the glycyrrhizin concentrations found to interfere with H5N1 replication and H5N1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels. Notably, although higher glycyrrhizin concentrations were needed to interfere with SARS coronavirus replication [22] than with H5N1 replication, beneficial results were reported in glycyrrhizin (SNMC)-treated SARS patients in comparison to SARS patients who did not receive glycyrrhizin [23] . Notably, investigation of different glycyrrhizin derivatives against SARS coronavirus led to the identification of compounds with enhanced antiviral activity [53] .", "Notably, investigation of different glycyrrhizin derivatives against SARS coronavirus led to the identification of compounds with enhanced antiviral activity [53] . Therefore, glycyrrhizin might also serve as lead structure for the development of novel anti-influenza drugs. Experimental results suggested that glycyrrhizin might be able to affect seasonal influenza A virus disease by antiviral and immunomodulatory effects [26, 27] .", "Experimental results suggested that glycyrrhizin might be able to affect seasonal influenza A virus disease by antiviral and immunomodulatory effects [26, 27] . Mice were prevented from lethal H2N2 infection by glycyrrhizin although no influence on virus replication was detected. The mechanism was suggested to be induction of interferon-c in T-cells by glycyrrhizin [54] .", "The mechanism was suggested to be induction of interferon-c in T-cells by glycyrrhizin [54] . Moreover, glycyrrhizin was shown to influence seasonal influenza A virus replication through interaction with the cell membrane [25, 28] . However, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period.", "However, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period. Since glycyrrhizin addition during the adsorption period did not influence H5N1 replication in our experiments it appears not likely that membrane effects contribute to anti-H5N1 effects detected here in lower concentrations. Our results rather suggest that glycyrrhizin interferes with H5N1-induced oxidative stress.", "Our results rather suggest that glycyrrhizin interferes with H5N1-induced oxidative stress. Influenza A virus (including H5N1) infection induces ROS formation. Antioxidants were found to inhibit influenza A virus replication and influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] .", "Antioxidants were found to inhibit influenza A virus replication and influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] . Here, glycyrrhizin interfered with H5N1-induced activation of NFkB, p38, and JNK representing redox-sensitive signalling events [48] [49] [50] [51] involved in influenza A virus replication and influenza A virusinduced cellular cytokine/chemokine production [34, [43] [44] [45] [46] 55] . Glycyrrhizin 50 mg/ml significantly reduced H5N1-induced activation of NFkB.", "Glycyrrhizin 50 mg/ml significantly reduced H5N1-induced activation of NFkB. In addition, glycyrrhizin concentrations as low as 25 mg/ml effectively interfered with H5N1-induced ROS formation and with phosphorylation of the redox-sensitive MAPKs p38 and JNK. In our model, activation of p38 appears to be critical for H5N1-associated redox signalling since p38 inhibition had been shown before to mimick effects of the antioxidant N-acetyl-cysteine (NAC) [34] .", "In our model, activation of p38 appears to be critical for H5N1-associated redox signalling since p38 inhibition had been shown before to mimick effects of the antioxidant N-acetyl-cysteine (NAC) [34] . Interestingly and in contrast to glycyrrhizin, NAC failed to inhibit H5N1 replication or H5N1-induced cytokine/chemokine expression in therapeutically relevant concentrations. Glycyrrhizin diminished H5N1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with H5N1 replication although redox-sensitive signalling pathways have been described to be involved in both processes.", "Glycyrrhizin diminished H5N1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with H5N1 replication although redox-sensitive signalling pathways have been described to be involved in both processes. Therefore, H5N1-induced proinflammatory gene expression appears to be more sensitive to inhibition of ROS formation than H5N1 replication. Indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] .", "Indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] . In a previous report, we could show that similar glycyrrhizin concentrations like those investigated here interfered with H5N1-induced pro-inflammatory gene expression but not with H5N1 replication in human monocyte-derived macrophages [57] . In addition, other immunomodulatory treatment regimens that did not influence H5N1 replication reduced mortality in H5N1-infected mice [31, 58] .", "In addition, other immunomodulatory treatment regimens that did not influence H5N1 replication reduced mortality in H5N1-infected mice [31, 58] . Therefore, glycyrrhizin represents a potential additional treatment option that interfers with both H5N1 replication and H5N1induced expression of pro-inflammatory cytokines in lung cells. Interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8 + T-lymphocytes.", "Interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8 + T-lymphocytes. Global immunosuppressants like corticosteroids failed to protect from lethal influenza virus infection [59] . Moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper NK cell cytolytic activity [60] .", "Moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper NK cell cytolytic activity [60] . In this context, glycyrrhizin had already been shown not to affect natural killer cell activity in the concentrations used here [57] . In conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation SNMC) interfere with highly pathogenic H5N1 influenza A virus replication and H5N1-induced proinflammatory gene expression at least in part through interference with H5N1-induced ROS formation and in turn reduced activation of p38, JNK, and NFkB in lung cells.", "In conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation SNMC) interfere with highly pathogenic H5N1 influenza A virus replication and H5N1-induced proinflammatory gene expression at least in part through interference with H5N1-induced ROS formation and in turn reduced activation of p38, JNK, and NFkB in lung cells. Since we used the clinical formulation SNMC effects of other ingredients like glycin or cystein cannot be excluded. Vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza A virus pandemic [61] .", "Vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza A virus pandemic [61] . Anti-inflammatory and immunomodulatory agents are considered to be important candidates as constituents of anti-influenza treatment strategies that may save lives in an influenza pandemic situation [61] . Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1-caused disease." ]

[ "Glycyrrhizin is known to exert antiviral and anti-inflammatory effects. Here, the effects of an approved parenteral glycyrrhizin preparation (Stronger Neo-Minophafen C) were investigated on highly pathogenic influenza A H5N1 virus replication, H5N1-induced apoptosis, and H5N1-induced pro-inflammatory responses in lung epithelial (A549) cells. Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher).", "Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher). Glycyrrhizin also diminished monocyte migration towards supernatants of H5N1-infected A549 cells. The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen species and (in turn) reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication.", "The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen species and (in turn) reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication. Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease. Text: Highly pathogenic H5N1 influenza A viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] .", "Text: Highly pathogenic H5N1 influenza A viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] . At least for the first wave of an H5N1 pandemic, no sufficient amounts of adequate vaccines will be available [1] [2] [3] [4] [6] [7] [8] . Therefore, antiviral therapy for influenza A viruses including highly pathogenic H5N1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] .", "Therefore, antiviral therapy for influenza A viruses including highly pathogenic H5N1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] . The neuraminidase inhibitors oseltamivir and zanamivir as well as the adamantanes amantadin and rimantadin that interfere with the influenza M2 protein are licensed for the treament of influenza [1] [2] [3] [4] 6] . However, the use of both drug classes is limited by the emergence of resistant virus strains.", "However, the use of both drug classes is limited by the emergence of resistant virus strains. In seasonal influenza strains, the majority of H3N2 viruses and a great proportion of H1N1 viruses in humans are now considered to be amantadine-and rimantadine-resistant [10] [11] [12] [13] . Moreover, a drastic increase in oseltamivir-resistant H1N1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] .", "Moreover, a drastic increase in oseltamivir-resistant H1N1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] . Preliminary data from the United States predict a further rise for the 2008/2009 season, possibly resulting in more than 90% of the circulating H1N1 strains to be oseltamivir resistant [14] . H5N1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza A virus strains and treatment-resistant H5N1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] .", "H5N1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza A virus strains and treatment-resistant H5N1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] . More-over, parenteral agents for the treatment of seriously ill patients are missing. Glycyrrhizin, a triterpene saponine, is a constituent of licorice root.", "Glycyrrhizin, a triterpene saponine, is a constituent of licorice root. It has been found to interfere with replication and/or cytopathogenic effect (CPE) induction of many viruses including respiratory viruses such as respiratory syncytial virus, SARS coronavirus, HIV, and influenza viruses [22] [23] [24] [25] [26] [27] [28] . Moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] .", "Moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] . The severity of human H5N1 disease has been associated with hypercytokinaemia (''cytokine storm'') [29, 30] . Delayed antiviral plus immunomodulator treatment reduced H5N1-induced mortality in mice [31] .", "Delayed antiviral plus immunomodulator treatment reduced H5N1-induced mortality in mice [31] . Therefore, antiinflammatory and immunomodulatory effects exerted by glycyrrhizin may be beneficial for treatment of H5N1. Also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza A virus replication and virus-induced pro-inflammatory responses [32] [33] [34] .", "Also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza A virus replication and virus-induced pro-inflammatory responses [32] [33] [34] . Stronger Neo-Minophagen C (SNMC) is a glycyrrhizin preparation (available as tablets or parenteral formulation) that is approved in Japan for the treatment of chronic hepatic diseases and is marketed in Japan, China, Korea, Taiwan, Indonesia, India, and Mongolia. Here, we investigated the influence of SNMC on H5N1 replication, on H5N1-induced cytokine expression, on H5N1-induced cellular oxidative stress, and on critical H5N1-induced cellular signalling events in human pneumocytes (A549 cell line).", "Here, we investigated the influence of SNMC on H5N1 replication, on H5N1-induced cytokine expression, on H5N1-induced cellular oxidative stress, and on critical H5N1-induced cellular signalling events in human pneumocytes (A549 cell line). Glycyrrhizin (Stronger Neo Minophagen C) was obtained from Minophagen Pharmaceuticals Co., Ltd. (Tokyo, Japan). The influenza strain A/Vietnam/1203/04 (H5N1) was received from the WHO Influenza Centre (National Institute for Medical Research, London, UK).", "The influenza strain A/Vietnam/1203/04 (H5N1) was received from the WHO Influenza Centre (National Institute for Medical Research, London, UK). The H5N1 influenza strain A/Thailand/ 1(Kan-1)/04 was obtained from Prof. Pilaipan Puthavathana (Mahidol University, Bangkok, Thailand). Virus stocks were prepared by infecting Vero cells (African green monkey kidney; ATCC, Manassas, VA) and aliquots were stored at 280uC.", "Virus stocks were prepared by infecting Vero cells (African green monkey kidney; ATCC, Manassas, VA) and aliquots were stored at 280uC. Virus titres were determined as 50% tissue culture infectious dose (TCID 50 /ml) in confluent Vero cells in 96-well microtiter plates. A549 cells (human lung carcinoma; ATCC: CCL-185, obtained from LGC Standards GmbH, Wesel, Germany) were grown at 37uC in minimal essential medium (MEM) supplemented with 10% FBS, 100 IU/ml of penicillin and 100 mg/ml streptomycin.", "A549 cells (human lung carcinoma; ATCC: CCL-185, obtained from LGC Standards GmbH, Wesel, Germany) were grown at 37uC in minimal essential medium (MEM) supplemented with 10% FBS, 100 IU/ml of penicillin and 100 mg/ml streptomycin. Human monocytes were isolated from buffy coats of healthy donors, obtained from Institute of Transfusion Medicine and Immune Haematology, German Red Cross Blood Donor Center, Johann Wolfgang Goethe-University, Frankfurt am Main. After centrifugation on Ficoll (Biocoll)-Hypaque density gradient (Biochrom AG, Berlin, Germany), mononuclear cells were collected from the interface and washed with PBS.", "After centrifugation on Ficoll (Biocoll)-Hypaque density gradient (Biochrom AG, Berlin, Germany), mononuclear cells were collected from the interface and washed with PBS. Then, monocytes were isolated using magnetically labeled CD14 MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) following the manufacturer's instructions. Monocytes were cultivated in IMDM supplemented with 10% pooled human serum, 100 IU/ml of penicillin, and 100 mg/ml streptomycin.", "Monocytes were cultivated in IMDM supplemented with 10% pooled human serum, 100 IU/ml of penicillin, and 100 mg/ml streptomycin. The cellular viability was assessed on confluent cell layers with CellTiter-GloH Luminescent Cell Viability Assay (Promega GmbH, Mannheim, Germany) according to the manufacturers' protocol. Cell viability was expressed as percentage of non-treated control.", "Cell viability was expressed as percentage of non-treated control. To determine intracellular NP localisation, H5N1-infected A549 were fixed 8 hours p.i. for 15 min with ice-cold acetone/ methanol (40:60, Mallinckrodt Baker B.V., Deventer, The Netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in PBS) directed against the influenza A virus nucleoprotein (NP) (Millipore, Molsheim, France).", "for 15 min with ice-cold acetone/ methanol (40:60, Mallinckrodt Baker B.V., Deventer, The Netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in PBS) directed against the influenza A virus nucleoprotein (NP) (Millipore, Molsheim, France). An Alexa Fluor 488 goat anti-mouse IgG (H&L) (Invitrogen, Eugene, Oregon, USA) was used (1 h incubation, 1:1000 in PBS) as secondary antibody. Nuclei were stained using 49,6-diamidino-2phenylindole (DAPI) (Sigma-Aldrich Chemie GmbH, Munich, Germany).", "Nuclei were stained using 49,6-diamidino-2phenylindole (DAPI) (Sigma-Aldrich Chemie GmbH, Munich, Germany). Fluorescence was visualised using Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany). For flow cytometric analysis, the same antibodies were used. The cytopathogenic effect (CPE) reduction assay was performed as described before [34] .", "The cytopathogenic effect (CPE) reduction assay was performed as described before [34] . Confluent A549 cell monolayers grown in 96-well microtitre plates were infected with influenza A strains at the indicated multiplicities of infection (MOIs). After a one hour adsorption period, cells were washed to remove non-detached virus.", "After a one hour adsorption period, cells were washed to remove non-detached virus. The virus-induced CPE was recorded at 24 h post infection (p.i.). Unless otherwise stated, A549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period.", "Unless otherwise stated, A549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period. For time-ofaddition experiments, glycyrrhizin was added exclusively during the 1 h pre-incubation period, exclusively during the 1 h adsorption period, or after exclusively after the wash-out of input virus. Total RNA was isolated from cell cultures using TRI reagent (Sigma-Aldrich, Munich, Germany).", "Total RNA was isolated from cell cultures using TRI reagent (Sigma-Aldrich, Munich, Germany). Real time PCR for H5 was performed using described methods [35] . The following primers were used: sense 59 acg tat gac tac ccg cag tat tca g 39; antisense 59 aga cca gcy acc atg att gc 39; probe 6-FAM-tca aca gtg gcg agt tcc cta gca-TAMRA.", "The following primers were used: sense 59 acg tat gac tac ccg cag tat tca g 39; antisense 59 aga cca gcy acc atg att gc 39; probe 6-FAM-tca aca gtg gcg agt tcc cta gca-TAMRA. The fraction of cells with fractional DNA content (''sub-G1'' cell subpopulation) indicates cytotoxicity. Sub-G1 cells are considered to be dead (usually apoptotic) cells.", "Sub-G1 cells are considered to be dead (usually apoptotic) cells. Cells were fixed with 70% ethanol for two hours at 220uC. The cellular DNA was stained using propidium iodide (20 mg/ml) and analysed by flow cytometry (FacsCalibur, BD Biosciences, Heidelberg, Germany).", "The cellular DNA was stained using propidium iodide (20 mg/ml) and analysed by flow cytometry (FacsCalibur, BD Biosciences, Heidelberg, Germany). Caspase activation was measured using the Caspase-Glo 8, 9, or 3/7 Assays (Promega, Mannheim, Germany) following the manufacturer's instructions. Cell culture supernatants were collected and frozen at 280uC.", "Cell culture supernatants were collected and frozen at 280uC. Cytokines/chemokines were quantified by specific ELISA Duo Sets (R&D Systems GmbH, Wiesbaden, Germany) following the manufacturer's instructions. NFkB activity was investigated in H5N1 (MOI 0.01)-infected cells by quantification of the NFkB subunits Rel A (p65) and NFkB1 (p50) from nuclear extracts using the TransAM TM transcription factor DNA-binding ELISAs (Active Motif, Rixensart, Belgium).", "NFkB activity was investigated in H5N1 (MOI 0.01)-infected cells by quantification of the NFkB subunits Rel A (p65) and NFkB1 (p50) from nuclear extracts using the TransAM TM transcription factor DNA-binding ELISAs (Active Motif, Rixensart, Belgium). Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction. Cell culture supernatants were investigated for chemotactic activity by measurement of the activity to induce monocyte migration through membrane inserts in 24-well plates (pore size 8 mm; BD Biosciences, Heidelberg, Germany).", "Cell culture supernatants were investigated for chemotactic activity by measurement of the activity to induce monocyte migration through membrane inserts in 24-well plates (pore size 8 mm; BD Biosciences, Heidelberg, Germany). Monocytes (1610 6 in 100 ml of IMDM with 10% pooled human serum) were added into the cell culture inserts (upper chamber) and cell culture supernatants (300 ml), were added to the lower chamber of the well. After a 48 h incubation period, cells were fixed with 4% paraformaldehyde and permeabilised with PBS containing 0.3% Tritron X-100.", "After a 48 h incubation period, cells were fixed with 4% paraformaldehyde and permeabilised with PBS containing 0.3% Tritron X-100. Then, nuclei were stained with 49,6-diamidino-2phenylindole (DAPI). The upper side of the membrane was wiped with a wet swab to remove the cells, while the lower side of the membrane was rinsed with PBS.", "The upper side of the membrane was wiped with a wet swab to remove the cells, while the lower side of the membrane was rinsed with PBS. The number of cells at the lower side of each membrane was quantified by counting of cells from three randomly chosen sections (3.7 mm 2 ) using an Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany). Cells were lysed in Triton X-sample buffer and separated by SDS-PAGE.", "Cells were lysed in Triton X-sample buffer and separated by SDS-PAGE. Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction. Proteins were detected using specific antibodies against bactin (Sigma-Aldrich Chemie GmbH, Munich, Germany), JNK, phosphorylated JNK, p38, or phosphorylated p38, (all purchased from New England Biolabs GmbH, Frankfurt am Main, Germany) and were visualised by enhanced chemiluminescence using a commercially available kit (Amersham, Freiburg, Germany).", "Proteins were detected using specific antibodies against bactin (Sigma-Aldrich Chemie GmbH, Munich, Germany), JNK, phosphorylated JNK, p38, or phosphorylated p38, (all purchased from New England Biolabs GmbH, Frankfurt am Main, Germany) and were visualised by enhanced chemiluminescence using a commercially available kit (Amersham, Freiburg, Germany). Reactive oxygen species (ROS) were detected using the Image-iT LIVE Green Reactive Oxygen Species Kit (Molecular Probes, distributed by Invitrogen, Karlsruhe, Germany). Two groups were compared by t-test.", "Two groups were compared by t-test. More groups were compared by ANOVA with subsequent Student-Newman-Keuls test. The A549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type II pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g.", "The A549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type II pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g. 6,37,38]. If not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period.", "If not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period. Glycyrrhizin 200 mg/ml (the maximum tested concentration) did not affect A549 cell viability (data not shown) but clearly decreased CPE formation in A549 cells infected with the H5N1 influenza strain A/Thailand/1(Kan-1)/04 at MOIs of 0.01, 0.1 or 1 ( Figure 1A ). Similar results were obtained in A549 cells infected with strain A/Vietnam/1203/04 (H5N1) (Suppl.", "Similar results were obtained in A549 cells infected with strain A/Vietnam/1203/04 (H5N1) (Suppl. Figure 1A) . Staining of A549 cells for influenza A nucleoprotein 24 h after infection with strain H5N1 A/Thailand/1(Kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza A nucleoprotein positive cells ( Figure 1B) .", "Staining of A549 cells for influenza A nucleoprotein 24 h after infection with strain H5N1 A/Thailand/1(Kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza A nucleoprotein positive cells ( Figure 1B) . To examine the influence of glycyrrhizin on virus progeny, A549 cells were infected with the H5N1 influenza strain A/ Thailand/1(Kan-1)/04 at MOI 0.01 or MOI 1 and infectious virus titres were determined 24 h post infection ( Figure 1C ). While glycyrrhizin in concentrations up to 50 mg/ml did not affect H5N1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (MOI 0.01: 13-fold reduction, MOI 1: 10-fold reduction).", "While glycyrrhizin in concentrations up to 50 mg/ml did not affect H5N1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (MOI 0.01: 13-fold reduction, MOI 1: 10-fold reduction). Next, influence of glycyrrhizin on H5N1 replication was confirmed by the detection of viral (H5) RNA using quantitative PCR. Only glycyrrhizin concentrations $100 mg/ml significantly reduced Figure 1B) or H5N1 A/Vietnam/1203/04-infected (Suppl.", "Only glycyrrhizin concentrations $100 mg/ml significantly reduced Figure 1B) or H5N1 A/Vietnam/1203/04-infected (Suppl. Figure 1C ) A549 cells (MOI 0.01) 24 h post infection. Time-of-addition experiments revealed that maximal effects were achieved when glycyrrhizin was continuously present starting with a 1 h pre-incubation period ( Figure 1D ).", "Time-of-addition experiments revealed that maximal effects were achieved when glycyrrhizin was continuously present starting with a 1 h pre-incubation period ( Figure 1D ). Addition of glycyrrhizin post infection showed reduced antiviral effects while pre-incubation alone or glycyrrhizin addition during the adsorption period did not significantly affect H5N1 replication. For investigation of H5N1-induced cytokine expression, five pro-inflammatory genes were chosen that had been correlated to severity of influenza disease: CXCL10 (also known as interferon-cinducible protein 10, IP-10), interleukin 6 (IL6), interleukin 8, (IL8; also known as CXCL8), CCL2 (also known as monocyte chemoattractant protein 1, MCP-1), and CCL5 (also known as RANTES).", "For investigation of H5N1-induced cytokine expression, five pro-inflammatory genes were chosen that had been correlated to severity of influenza disease: CXCL10 (also known as interferon-cinducible protein 10, IP-10), interleukin 6 (IL6), interleukin 8, (IL8; also known as CXCL8), CCL2 (also known as monocyte chemoattractant protein 1, MCP-1), and CCL5 (also known as RANTES). A549 cells were infected with H5N1 A/Thailand/ 1(Kan-1)/04 or H5N1 A/Vietnam/1203/04 at MOI 0.01, 0.1, or 1. Glycyrrhizin treatment was performed with 25, 50, 100, or 200 mg/ml.", "Glycyrrhizin treatment was performed with 25, 50, 100, or 200 mg/ml. Cytokine expression was detected 24 h post infection by ELISA. Glycyrrhizin did not affect cytokine expression of noninfected cells (data not shown) but inhibited expression of all cytokines investigated in H5N1-infected cells in a dose-dependent manner (Figure 2, Figure 3A ).", "Glycyrrhizin did not affect cytokine expression of noninfected cells (data not shown) but inhibited expression of all cytokines investigated in H5N1-infected cells in a dose-dependent manner (Figure 2, Figure 3A ). Effects were more pronounced at lower MOIs. Notably, expression of all cytokines except IL8 was significantly inhibited after treatment with glycyrrhizin 50 mg/ml Figure 3A ) although these glycyrrhizin concentrations had no effect on H5N1 replication in A549 cells (Figure 1, Figure S1 ).", "Notably, expression of all cytokines except IL8 was significantly inhibited after treatment with glycyrrhizin 50 mg/ml Figure 3A ) although these glycyrrhizin concentrations had no effect on H5N1 replication in A549 cells (Figure 1, Figure S1 ). Cytokine expression by influenza A virus-infected respiratory cells causes recruitment of peripheral blood monocytes into the lungs of patients where they differentiate to macrophages which are thought to contribute to influenza A virus pathogenicity [5, 39] . In a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.1)-infected A549 cells through 8 mm filters.", "In a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.1)-infected A549 cells through 8 mm filters. Monocyte migration towards supernatants of H5N1-infected cells was strongly increased relative to migration towards supernatants of non-infected cells. Treatment of H5N1- infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( Figure 3B ).", "Treatment of H5N1- infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( Figure 3B ). Influenza viruses including H5N1 have been shown to induce caspase-dependent apoptosis in airway cells and this apoptosis has been correlated to the virus pathogenicity [40, 41] . Glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( Figure 4A-C) .", "Glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( Figure 4A-C) . Glycyrrhizin concentrations $100 mg/ml inhibited H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.01)-induced activation of the initiator caspases 8 and 9 as well as of the effector caspases 3/7 in A549 cells as determined 24 h post infection ( Figure 4A-C) . Lower glycyrrhizin concentrations did not affect H5N1-induced apoptosis.", "Lower glycyrrhizin concentrations did not affect H5N1-induced apoptosis. The detection of cells in sub-G1 phase resulted in similar findings ( Figure 4D ). Substances that inhibit H5N1-induced caspase 3 activation including caspase 3 inhibitors cause nuclear retention of RNP complexes [34, 42] .", "Substances that inhibit H5N1-induced caspase 3 activation including caspase 3 inhibitors cause nuclear retention of RNP complexes [34, 42] . In accordance, glycyrrhizin also interfered with nuclear export RNP at MOI 1 ( Figure S2 ). Similar results were obtained in MOI 0.01 H5N1 A/Thailand/1(Kan-1)/04infected cells ( Figure S3 ).", "Similar results were obtained in MOI 0.01 H5N1 A/Thailand/1(Kan-1)/04infected cells ( Figure S3 ). Influence of glycyrrhizin on H5N1-induced activation of nuclear factor kB (NFkB), p38, and on H5N1-induced cellular reactive oxygen species (ROS) formation Activation of NFkB, p38, and JNK have been associated with influenza A virus replication and virus-induced pro-inflammatory gene expression [34, [43] [44] [45] [46] [47] . While glycyrrhizin did not influence NFkB activity in non-infected A549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited NFkB activation in H5N1-infected cells ( Figure 5A ).", "While glycyrrhizin did not influence NFkB activity in non-infected A549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited NFkB activation in H5N1-infected cells ( Figure 5A ). Moreover, glycyrrhizin inhibited H5N1-induced phosphorylation of the MAPKs p38 and JNK ( Figure 5B ). In addition to their roles during influenza A virus replication and virus-induced cytokine/chemokine expression, NFkB, p38, and JNK are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] .", "In addition to their roles during influenza A virus replication and virus-induced cytokine/chemokine expression, NFkB, p38, and JNK are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] . Antioxidants had been already found to interfere with influenza A virus-induced signalling through NFkB, p38, and JNK, with influenza A virus replication, and with influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] . Since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with H5N1-induced ROS formation.", "Since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with H5N1-induced ROS formation. Indeed glycyrrhizin exerted clear antioxidative effects in H5N1 (MOI 0.01)-infected cells ( Figure 5C ) causing significant reduction of ROS formation already at a concentration of 25 mg/ml ( Figure 5D ). Here, we show that glycyrrhizin inhibits the replication of highly pathogenic H5N1 influenza A virus, H5N1-induced apoptosis, and H5N1-induced expression of pro-inflammatory cytokines in lung-derived A549 cells.", "Here, we show that glycyrrhizin inhibits the replication of highly pathogenic H5N1 influenza A virus, H5N1-induced apoptosis, and H5N1-induced expression of pro-inflammatory cytokines in lung-derived A549 cells. After intravenous administration, achievable plasma concentrations of glycyrrhizin have been described to be about 100 mg/ml [52] . Therefore, the glycyrrhizin concentrations found to interfere with H5N1 replication and H5N1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels.", "Therefore, the glycyrrhizin concentrations found to interfere with H5N1 replication and H5N1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels. Notably, although higher glycyrrhizin concentrations were needed to interfere with SARS coronavirus replication [22] than with H5N1 replication, beneficial results were reported in glycyrrhizin (SNMC)-treated SARS patients in comparison to SARS patients who did not receive glycyrrhizin [23] . Notably, investigation of different glycyrrhizin derivatives against SARS coronavirus led to the identification of compounds with enhanced antiviral activity [53] .", "Notably, investigation of different glycyrrhizin derivatives against SARS coronavirus led to the identification of compounds with enhanced antiviral activity [53] . Therefore, glycyrrhizin might also serve as lead structure for the development of novel anti-influenza drugs. Experimental results suggested that glycyrrhizin might be able to affect seasonal influenza A virus disease by antiviral and immunomodulatory effects [26, 27] .", "Experimental results suggested that glycyrrhizin might be able to affect seasonal influenza A virus disease by antiviral and immunomodulatory effects [26, 27] . Mice were prevented from lethal H2N2 infection by glycyrrhizin although no influence on virus replication was detected. The mechanism was suggested to be induction of interferon-c in T-cells by glycyrrhizin [54] .", "The mechanism was suggested to be induction of interferon-c in T-cells by glycyrrhizin [54] . Moreover, glycyrrhizin was shown to influence seasonal influenza A virus replication through interaction with the cell membrane [25, 28] . However, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period.", "However, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period. Since glycyrrhizin addition during the adsorption period did not influence H5N1 replication in our experiments it appears not likely that membrane effects contribute to anti-H5N1 effects detected here in lower concentrations. Our results rather suggest that glycyrrhizin interferes with H5N1-induced oxidative stress.", "Our results rather suggest that glycyrrhizin interferes with H5N1-induced oxidative stress. Influenza A virus (including H5N1) infection induces ROS formation. Antioxidants were found to inhibit influenza A virus replication and influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] .", "Antioxidants were found to inhibit influenza A virus replication and influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] . Here, glycyrrhizin interfered with H5N1-induced activation of NFkB, p38, and JNK representing redox-sensitive signalling events [48] [49] [50] [51] involved in influenza A virus replication and influenza A virusinduced cellular cytokine/chemokine production [34, [43] [44] [45] [46] 55] . Glycyrrhizin 50 mg/ml significantly reduced H5N1-induced activation of NFkB.", "Glycyrrhizin 50 mg/ml significantly reduced H5N1-induced activation of NFkB. In addition, glycyrrhizin concentrations as low as 25 mg/ml effectively interfered with H5N1-induced ROS formation and with phosphorylation of the redox-sensitive MAPKs p38 and JNK. In our model, activation of p38 appears to be critical for H5N1-associated redox signalling since p38 inhibition had been shown before to mimick effects of the antioxidant N-acetyl-cysteine (NAC) [34] .", "In our model, activation of p38 appears to be critical for H5N1-associated redox signalling since p38 inhibition had been shown before to mimick effects of the antioxidant N-acetyl-cysteine (NAC) [34] . Interestingly and in contrast to glycyrrhizin, NAC failed to inhibit H5N1 replication or H5N1-induced cytokine/chemokine expression in therapeutically relevant concentrations. Glycyrrhizin diminished H5N1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with H5N1 replication although redox-sensitive signalling pathways have been described to be involved in both processes.", "Glycyrrhizin diminished H5N1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with H5N1 replication although redox-sensitive signalling pathways have been described to be involved in both processes. Therefore, H5N1-induced proinflammatory gene expression appears to be more sensitive to inhibition of ROS formation than H5N1 replication. Indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] .", "Indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] . In a previous report, we could show that similar glycyrrhizin concentrations like those investigated here interfered with H5N1-induced pro-inflammatory gene expression but not with H5N1 replication in human monocyte-derived macrophages [57] . In addition, other immunomodulatory treatment regimens that did not influence H5N1 replication reduced mortality in H5N1-infected mice [31, 58] .", "In addition, other immunomodulatory treatment regimens that did not influence H5N1 replication reduced mortality in H5N1-infected mice [31, 58] . Therefore, glycyrrhizin represents a potential additional treatment option that interfers with both H5N1 replication and H5N1induced expression of pro-inflammatory cytokines in lung cells. Interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8 + T-lymphocytes.", "Interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8 + T-lymphocytes. Global immunosuppressants like corticosteroids failed to protect from lethal influenza virus infection [59] . Moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper NK cell cytolytic activity [60] .", "Moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper NK cell cytolytic activity [60] . In this context, glycyrrhizin had already been shown not to affect natural killer cell activity in the concentrations used here [57] . In conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation SNMC) interfere with highly pathogenic H5N1 influenza A virus replication and H5N1-induced proinflammatory gene expression at least in part through interference with H5N1-induced ROS formation and in turn reduced activation of p38, JNK, and NFkB in lung cells.", "In conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation SNMC) interfere with highly pathogenic H5N1 influenza A virus replication and H5N1-induced proinflammatory gene expression at least in part through interference with H5N1-induced ROS formation and in turn reduced activation of p38, JNK, and NFkB in lung cells. Since we used the clinical formulation SNMC effects of other ingredients like glycin or cystein cannot be excluded. Vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza A virus pandemic [61] .", "Vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza A virus pandemic [61] . Anti-inflammatory and immunomodulatory agents are considered to be important candidates as constituents of anti-influenza treatment strategies that may save lives in an influenza pandemic situation [61] . Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1-caused disease." ]

[ "Glycyrrhizin is known to exert antiviral and anti-inflammatory effects. Here, the effects of an approved parenteral glycyrrhizin preparation (Stronger Neo-Minophafen C) were investigated on highly pathogenic influenza A H5N1 virus replication, H5N1-induced apoptosis, and H5N1-induced pro-inflammatory responses in lung epithelial (A549) cells. Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher).", "Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher). Glycyrrhizin also diminished monocyte migration towards supernatants of H5N1-infected A549 cells. The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen species and (in turn) reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication.", "The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen species and (in turn) reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication. Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease. Text: Highly pathogenic H5N1 influenza A viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] .", "Text: Highly pathogenic H5N1 influenza A viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] . At least for the first wave of an H5N1 pandemic, no sufficient amounts of adequate vaccines will be available [1] [2] [3] [4] [6] [7] [8] . Therefore, antiviral therapy for influenza A viruses including highly pathogenic H5N1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] .", "Therefore, antiviral therapy for influenza A viruses including highly pathogenic H5N1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] . The neuraminidase inhibitors oseltamivir and zanamivir as well as the adamantanes amantadin and rimantadin that interfere with the influenza M2 protein are licensed for the treament of influenza [1] [2] [3] [4] 6] . However, the use of both drug classes is limited by the emergence of resistant virus strains.", "However, the use of both drug classes is limited by the emergence of resistant virus strains. In seasonal influenza strains, the majority of H3N2 viruses and a great proportion of H1N1 viruses in humans are now considered to be amantadine-and rimantadine-resistant [10] [11] [12] [13] . Moreover, a drastic increase in oseltamivir-resistant H1N1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] .", "Moreover, a drastic increase in oseltamivir-resistant H1N1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] . Preliminary data from the United States predict a further rise for the 2008/2009 season, possibly resulting in more than 90% of the circulating H1N1 strains to be oseltamivir resistant [14] . H5N1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza A virus strains and treatment-resistant H5N1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] .", "H5N1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza A virus strains and treatment-resistant H5N1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] . More-over, parenteral agents for the treatment of seriously ill patients are missing. Glycyrrhizin, a triterpene saponine, is a constituent of licorice root.", "Glycyrrhizin, a triterpene saponine, is a constituent of licorice root. It has been found to interfere with replication and/or cytopathogenic effect (CPE) induction of many viruses including respiratory viruses such as respiratory syncytial virus, SARS coronavirus, HIV, and influenza viruses [22] [23] [24] [25] [26] [27] [28] . Moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] .", "Moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] . The severity of human H5N1 disease has been associated with hypercytokinaemia (''cytokine storm'') [29, 30] . Delayed antiviral plus immunomodulator treatment reduced H5N1-induced mortality in mice [31] .", "Delayed antiviral plus immunomodulator treatment reduced H5N1-induced mortality in mice [31] . Therefore, antiinflammatory and immunomodulatory effects exerted by glycyrrhizin may be beneficial for treatment of H5N1. Also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza A virus replication and virus-induced pro-inflammatory responses [32] [33] [34] .", "Also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza A virus replication and virus-induced pro-inflammatory responses [32] [33] [34] . Stronger Neo-Minophagen C (SNMC) is a glycyrrhizin preparation (available as tablets or parenteral formulation) that is approved in Japan for the treatment of chronic hepatic diseases and is marketed in Japan, China, Korea, Taiwan, Indonesia, India, and Mongolia. Here, we investigated the influence of SNMC on H5N1 replication, on H5N1-induced cytokine expression, on H5N1-induced cellular oxidative stress, and on critical H5N1-induced cellular signalling events in human pneumocytes (A549 cell line).", "Here, we investigated the influence of SNMC on H5N1 replication, on H5N1-induced cytokine expression, on H5N1-induced cellular oxidative stress, and on critical H5N1-induced cellular signalling events in human pneumocytes (A549 cell line). Glycyrrhizin (Stronger Neo Minophagen C) was obtained from Minophagen Pharmaceuticals Co., Ltd. (Tokyo, Japan). The influenza strain A/Vietnam/1203/04 (H5N1) was received from the WHO Influenza Centre (National Institute for Medical Research, London, UK).", "The influenza strain A/Vietnam/1203/04 (H5N1) was received from the WHO Influenza Centre (National Institute for Medical Research, London, UK). The H5N1 influenza strain A/Thailand/ 1(Kan-1)/04 was obtained from Prof. Pilaipan Puthavathana (Mahidol University, Bangkok, Thailand). Virus stocks were prepared by infecting Vero cells (African green monkey kidney; ATCC, Manassas, VA) and aliquots were stored at 280uC.", "Virus stocks were prepared by infecting Vero cells (African green monkey kidney; ATCC, Manassas, VA) and aliquots were stored at 280uC. Virus titres were determined as 50% tissue culture infectious dose (TCID 50 /ml) in confluent Vero cells in 96-well microtiter plates. A549 cells (human lung carcinoma; ATCC: CCL-185, obtained from LGC Standards GmbH, Wesel, Germany) were grown at 37uC in minimal essential medium (MEM) supplemented with 10% FBS, 100 IU/ml of penicillin and 100 mg/ml streptomycin.", "A549 cells (human lung carcinoma; ATCC: CCL-185, obtained from LGC Standards GmbH, Wesel, Germany) were grown at 37uC in minimal essential medium (MEM) supplemented with 10% FBS, 100 IU/ml of penicillin and 100 mg/ml streptomycin. Human monocytes were isolated from buffy coats of healthy donors, obtained from Institute of Transfusion Medicine and Immune Haematology, German Red Cross Blood Donor Center, Johann Wolfgang Goethe-University, Frankfurt am Main. After centrifugation on Ficoll (Biocoll)-Hypaque density gradient (Biochrom AG, Berlin, Germany), mononuclear cells were collected from the interface and washed with PBS.", "After centrifugation on Ficoll (Biocoll)-Hypaque density gradient (Biochrom AG, Berlin, Germany), mononuclear cells were collected from the interface and washed with PBS. Then, monocytes were isolated using magnetically labeled CD14 MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) following the manufacturer's instructions. Monocytes were cultivated in IMDM supplemented with 10% pooled human serum, 100 IU/ml of penicillin, and 100 mg/ml streptomycin.", "Monocytes were cultivated in IMDM supplemented with 10% pooled human serum, 100 IU/ml of penicillin, and 100 mg/ml streptomycin. The cellular viability was assessed on confluent cell layers with CellTiter-GloH Luminescent Cell Viability Assay (Promega GmbH, Mannheim, Germany) according to the manufacturers' protocol. Cell viability was expressed as percentage of non-treated control.", "Cell viability was expressed as percentage of non-treated control. To determine intracellular NP localisation, H5N1-infected A549 were fixed 8 hours p.i. for 15 min with ice-cold acetone/ methanol (40:60, Mallinckrodt Baker B.V., Deventer, The Netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in PBS) directed against the influenza A virus nucleoprotein (NP) (Millipore, Molsheim, France).", "for 15 min with ice-cold acetone/ methanol (40:60, Mallinckrodt Baker B.V., Deventer, The Netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in PBS) directed against the influenza A virus nucleoprotein (NP) (Millipore, Molsheim, France). An Alexa Fluor 488 goat anti-mouse IgG (H&L) (Invitrogen, Eugene, Oregon, USA) was used (1 h incubation, 1:1000 in PBS) as secondary antibody. Nuclei were stained using 49,6-diamidino-2phenylindole (DAPI) (Sigma-Aldrich Chemie GmbH, Munich, Germany).", "Nuclei were stained using 49,6-diamidino-2phenylindole (DAPI) (Sigma-Aldrich Chemie GmbH, Munich, Germany). Fluorescence was visualised using Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany). For flow cytometric analysis, the same antibodies were used. The cytopathogenic effect (CPE) reduction assay was performed as described before [34] .", "The cytopathogenic effect (CPE) reduction assay was performed as described before [34] . Confluent A549 cell monolayers grown in 96-well microtitre plates were infected with influenza A strains at the indicated multiplicities of infection (MOIs). After a one hour adsorption period, cells were washed to remove non-detached virus.", "After a one hour adsorption period, cells were washed to remove non-detached virus. The virus-induced CPE was recorded at 24 h post infection (p.i.). Unless otherwise stated, A549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period.", "Unless otherwise stated, A549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period. For time-ofaddition experiments, glycyrrhizin was added exclusively during the 1 h pre-incubation period, exclusively during the 1 h adsorption period, or after exclusively after the wash-out of input virus. Total RNA was isolated from cell cultures using TRI reagent (Sigma-Aldrich, Munich, Germany).", "Total RNA was isolated from cell cultures using TRI reagent (Sigma-Aldrich, Munich, Germany). Real time PCR for H5 was performed using described methods [35] . The following primers were used: sense 59 acg tat gac tac ccg cag tat tca g 39; antisense 59 aga cca gcy acc atg att gc 39; probe 6-FAM-tca aca gtg gcg agt tcc cta gca-TAMRA.", "The following primers were used: sense 59 acg tat gac tac ccg cag tat tca g 39; antisense 59 aga cca gcy acc atg att gc 39; probe 6-FAM-tca aca gtg gcg agt tcc cta gca-TAMRA. The fraction of cells with fractional DNA content (''sub-G1'' cell subpopulation) indicates cytotoxicity. Sub-G1 cells are considered to be dead (usually apoptotic) cells.", "Sub-G1 cells are considered to be dead (usually apoptotic) cells. Cells were fixed with 70% ethanol for two hours at 220uC. The cellular DNA was stained using propidium iodide (20 mg/ml) and analysed by flow cytometry (FacsCalibur, BD Biosciences, Heidelberg, Germany).", "The cellular DNA was stained using propidium iodide (20 mg/ml) and analysed by flow cytometry (FacsCalibur, BD Biosciences, Heidelberg, Germany). Caspase activation was measured using the Caspase-Glo 8, 9, or 3/7 Assays (Promega, Mannheim, Germany) following the manufacturer's instructions. Cell culture supernatants were collected and frozen at 280uC.", "Cell culture supernatants were collected and frozen at 280uC. Cytokines/chemokines were quantified by specific ELISA Duo Sets (R&D Systems GmbH, Wiesbaden, Germany) following the manufacturer's instructions. NFkB activity was investigated in H5N1 (MOI 0.01)-infected cells by quantification of the NFkB subunits Rel A (p65) and NFkB1 (p50) from nuclear extracts using the TransAM TM transcription factor DNA-binding ELISAs (Active Motif, Rixensart, Belgium).", "NFkB activity was investigated in H5N1 (MOI 0.01)-infected cells by quantification of the NFkB subunits Rel A (p65) and NFkB1 (p50) from nuclear extracts using the TransAM TM transcription factor DNA-binding ELISAs (Active Motif, Rixensart, Belgium). Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction. Cell culture supernatants were investigated for chemotactic activity by measurement of the activity to induce monocyte migration through membrane inserts in 24-well plates (pore size 8 mm; BD Biosciences, Heidelberg, Germany).", "Cell culture supernatants were investigated for chemotactic activity by measurement of the activity to induce monocyte migration through membrane inserts in 24-well plates (pore size 8 mm; BD Biosciences, Heidelberg, Germany). Monocytes (1610 6 in 100 ml of IMDM with 10% pooled human serum) were added into the cell culture inserts (upper chamber) and cell culture supernatants (300 ml), were added to the lower chamber of the well. After a 48 h incubation period, cells were fixed with 4% paraformaldehyde and permeabilised with PBS containing 0.3% Tritron X-100.", "After a 48 h incubation period, cells were fixed with 4% paraformaldehyde and permeabilised with PBS containing 0.3% Tritron X-100. Then, nuclei were stained with 49,6-diamidino-2phenylindole (DAPI). The upper side of the membrane was wiped with a wet swab to remove the cells, while the lower side of the membrane was rinsed with PBS.", "The upper side of the membrane was wiped with a wet swab to remove the cells, while the lower side of the membrane was rinsed with PBS. The number of cells at the lower side of each membrane was quantified by counting of cells from three randomly chosen sections (3.7 mm 2 ) using an Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany). Cells were lysed in Triton X-sample buffer and separated by SDS-PAGE.", "Cells were lysed in Triton X-sample buffer and separated by SDS-PAGE. Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction. Proteins were detected using specific antibodies against bactin (Sigma-Aldrich Chemie GmbH, Munich, Germany), JNK, phosphorylated JNK, p38, or phosphorylated p38, (all purchased from New England Biolabs GmbH, Frankfurt am Main, Germany) and were visualised by enhanced chemiluminescence using a commercially available kit (Amersham, Freiburg, Germany).", "Proteins were detected using specific antibodies against bactin (Sigma-Aldrich Chemie GmbH, Munich, Germany), JNK, phosphorylated JNK, p38, or phosphorylated p38, (all purchased from New England Biolabs GmbH, Frankfurt am Main, Germany) and were visualised by enhanced chemiluminescence using a commercially available kit (Amersham, Freiburg, Germany). Reactive oxygen species (ROS) were detected using the Image-iT LIVE Green Reactive Oxygen Species Kit (Molecular Probes, distributed by Invitrogen, Karlsruhe, Germany). Two groups were compared by t-test.", "Two groups were compared by t-test. More groups were compared by ANOVA with subsequent Student-Newman-Keuls test. The A549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type II pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g.", "The A549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type II pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g. 6,37,38]. If not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period.", "If not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period. Glycyrrhizin 200 mg/ml (the maximum tested concentration) did not affect A549 cell viability (data not shown) but clearly decreased CPE formation in A549 cells infected with the H5N1 influenza strain A/Thailand/1(Kan-1)/04 at MOIs of 0.01, 0.1 or 1 ( Figure 1A ). Similar results were obtained in A549 cells infected with strain A/Vietnam/1203/04 (H5N1) (Suppl.", "Similar results were obtained in A549 cells infected with strain A/Vietnam/1203/04 (H5N1) (Suppl. Figure 1A) . Staining of A549 cells for influenza A nucleoprotein 24 h after infection with strain H5N1 A/Thailand/1(Kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza A nucleoprotein positive cells ( Figure 1B) .", "Staining of A549 cells for influenza A nucleoprotein 24 h after infection with strain H5N1 A/Thailand/1(Kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza A nucleoprotein positive cells ( Figure 1B) . To examine the influence of glycyrrhizin on virus progeny, A549 cells were infected with the H5N1 influenza strain A/ Thailand/1(Kan-1)/04 at MOI 0.01 or MOI 1 and infectious virus titres were determined 24 h post infection ( Figure 1C ). While glycyrrhizin in concentrations up to 50 mg/ml did not affect H5N1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (MOI 0.01: 13-fold reduction, MOI 1: 10-fold reduction).", "While glycyrrhizin in concentrations up to 50 mg/ml did not affect H5N1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (MOI 0.01: 13-fold reduction, MOI 1: 10-fold reduction). Next, influence of glycyrrhizin on H5N1 replication was confirmed by the detection of viral (H5) RNA using quantitative PCR. Only glycyrrhizin concentrations $100 mg/ml significantly reduced Figure 1B) or H5N1 A/Vietnam/1203/04-infected (Suppl.", "Only glycyrrhizin concentrations $100 mg/ml significantly reduced Figure 1B) or H5N1 A/Vietnam/1203/04-infected (Suppl. Figure 1C ) A549 cells (MOI 0.01) 24 h post infection. Time-of-addition experiments revealed that maximal effects were achieved when glycyrrhizin was continuously present starting with a 1 h pre-incubation period ( Figure 1D ).", "Time-of-addition experiments revealed that maximal effects were achieved when glycyrrhizin was continuously present starting with a 1 h pre-incubation period ( Figure 1D ). Addition of glycyrrhizin post infection showed reduced antiviral effects while pre-incubation alone or glycyrrhizin addition during the adsorption period did not significantly affect H5N1 replication. For investigation of H5N1-induced cytokine expression, five pro-inflammatory genes were chosen that had been correlated to severity of influenza disease: CXCL10 (also known as interferon-cinducible protein 10, IP-10), interleukin 6 (IL6), interleukin 8, (IL8; also known as CXCL8), CCL2 (also known as monocyte chemoattractant protein 1, MCP-1), and CCL5 (also known as RANTES).", "For investigation of H5N1-induced cytokine expression, five pro-inflammatory genes were chosen that had been correlated to severity of influenza disease: CXCL10 (also known as interferon-cinducible protein 10, IP-10), interleukin 6 (IL6), interleukin 8, (IL8; also known as CXCL8), CCL2 (also known as monocyte chemoattractant protein 1, MCP-1), and CCL5 (also known as RANTES). A549 cells were infected with H5N1 A/Thailand/ 1(Kan-1)/04 or H5N1 A/Vietnam/1203/04 at MOI 0.01, 0.1, or 1. Glycyrrhizin treatment was performed with 25, 50, 100, or 200 mg/ml.", "Glycyrrhizin treatment was performed with 25, 50, 100, or 200 mg/ml. Cytokine expression was detected 24 h post infection by ELISA. Glycyrrhizin did not affect cytokine expression of noninfected cells (data not shown) but inhibited expression of all cytokines investigated in H5N1-infected cells in a dose-dependent manner (Figure 2, Figure 3A ).", "Glycyrrhizin did not affect cytokine expression of noninfected cells (data not shown) but inhibited expression of all cytokines investigated in H5N1-infected cells in a dose-dependent manner (Figure 2, Figure 3A ). Effects were more pronounced at lower MOIs. Notably, expression of all cytokines except IL8 was significantly inhibited after treatment with glycyrrhizin 50 mg/ml Figure 3A ) although these glycyrrhizin concentrations had no effect on H5N1 replication in A549 cells (Figure 1, Figure S1 ).", "Notably, expression of all cytokines except IL8 was significantly inhibited after treatment with glycyrrhizin 50 mg/ml Figure 3A ) although these glycyrrhizin concentrations had no effect on H5N1 replication in A549 cells (Figure 1, Figure S1 ). Cytokine expression by influenza A virus-infected respiratory cells causes recruitment of peripheral blood monocytes into the lungs of patients where they differentiate to macrophages which are thought to contribute to influenza A virus pathogenicity [5, 39] . In a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.1)-infected A549 cells through 8 mm filters.", "In a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.1)-infected A549 cells through 8 mm filters. Monocyte migration towards supernatants of H5N1-infected cells was strongly increased relative to migration towards supernatants of non-infected cells. Treatment of H5N1- infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( Figure 3B ).", "Treatment of H5N1- infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( Figure 3B ). Influenza viruses including H5N1 have been shown to induce caspase-dependent apoptosis in airway cells and this apoptosis has been correlated to the virus pathogenicity [40, 41] . Glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( Figure 4A-C) .", "Glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( Figure 4A-C) . Glycyrrhizin concentrations $100 mg/ml inhibited H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.01)-induced activation of the initiator caspases 8 and 9 as well as of the effector caspases 3/7 in A549 cells as determined 24 h post infection ( Figure 4A-C) . Lower glycyrrhizin concentrations did not affect H5N1-induced apoptosis.", "Lower glycyrrhizin concentrations did not affect H5N1-induced apoptosis. The detection of cells in sub-G1 phase resulted in similar findings ( Figure 4D ). Substances that inhibit H5N1-induced caspase 3 activation including caspase 3 inhibitors cause nuclear retention of RNP complexes [34, 42] .", "Substances that inhibit H5N1-induced caspase 3 activation including caspase 3 inhibitors cause nuclear retention of RNP complexes [34, 42] . In accordance, glycyrrhizin also interfered with nuclear export RNP at MOI 1 ( Figure S2 ). Similar results were obtained in MOI 0.01 H5N1 A/Thailand/1(Kan-1)/04infected cells ( Figure S3 ).", "Similar results were obtained in MOI 0.01 H5N1 A/Thailand/1(Kan-1)/04infected cells ( Figure S3 ). Influence of glycyrrhizin on H5N1-induced activation of nuclear factor kB (NFkB), p38, and on H5N1-induced cellular reactive oxygen species (ROS) formation Activation of NFkB, p38, and JNK have been associated with influenza A virus replication and virus-induced pro-inflammatory gene expression [34, [43] [44] [45] [46] [47] . While glycyrrhizin did not influence NFkB activity in non-infected A549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited NFkB activation in H5N1-infected cells ( Figure 5A ).", "While glycyrrhizin did not influence NFkB activity in non-infected A549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited NFkB activation in H5N1-infected cells ( Figure 5A ). Moreover, glycyrrhizin inhibited H5N1-induced phosphorylation of the MAPKs p38 and JNK ( Figure 5B ). In addition to their roles during influenza A virus replication and virus-induced cytokine/chemokine expression, NFkB, p38, and JNK are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] .", "In addition to their roles during influenza A virus replication and virus-induced cytokine/chemokine expression, NFkB, p38, and JNK are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] . Antioxidants had been already found to interfere with influenza A virus-induced signalling through NFkB, p38, and JNK, with influenza A virus replication, and with influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] . Since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with H5N1-induced ROS formation.", "Since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with H5N1-induced ROS formation. Indeed glycyrrhizin exerted clear antioxidative effects in H5N1 (MOI 0.01)-infected cells ( Figure 5C ) causing significant reduction of ROS formation already at a concentration of 25 mg/ml ( Figure 5D ). Here, we show that glycyrrhizin inhibits the replication of highly pathogenic H5N1 influenza A virus, H5N1-induced apoptosis, and H5N1-induced expression of pro-inflammatory cytokines in lung-derived A549 cells.", "Here, we show that glycyrrhizin inhibits the replication of highly pathogenic H5N1 influenza A virus, H5N1-induced apoptosis, and H5N1-induced expression of pro-inflammatory cytokines in lung-derived A549 cells. After intravenous administration, achievable plasma concentrations of glycyrrhizin have been described to be about 100 mg/ml [52] . Therefore, the glycyrrhizin concentrations found to interfere with H5N1 replication and H5N1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels.", "Therefore, the glycyrrhizin concentrations found to interfere with H5N1 replication and H5N1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels. Notably, although higher glycyrrhizin concentrations were needed to interfere with SARS coronavirus replication [22] than with H5N1 replication, beneficial results were reported in glycyrrhizin (SNMC)-treated SARS patients in comparison to SARS patients who did not receive glycyrrhizin [23] . Notably, investigation of different glycyrrhizin derivatives against SARS coronavirus led to the identification of compounds with enhanced antiviral activity [53] .", "Notably, investigation of different glycyrrhizin derivatives against SARS coronavirus led to the identification of compounds with enhanced antiviral activity [53] . Therefore, glycyrrhizin might also serve as lead structure for the development of novel anti-influenza drugs. Experimental results suggested that glycyrrhizin might be able to affect seasonal influenza A virus disease by antiviral and immunomodulatory effects [26, 27] .", "Experimental results suggested that glycyrrhizin might be able to affect seasonal influenza A virus disease by antiviral and immunomodulatory effects [26, 27] . Mice were prevented from lethal H2N2 infection by glycyrrhizin although no influence on virus replication was detected. The mechanism was suggested to be induction of interferon-c in T-cells by glycyrrhizin [54] .", "The mechanism was suggested to be induction of interferon-c in T-cells by glycyrrhizin [54] . Moreover, glycyrrhizin was shown to influence seasonal influenza A virus replication through interaction with the cell membrane [25, 28] . However, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period.", "However, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period. Since glycyrrhizin addition during the adsorption period did not influence H5N1 replication in our experiments it appears not likely that membrane effects contribute to anti-H5N1 effects detected here in lower concentrations. Our results rather suggest that glycyrrhizin interferes with H5N1-induced oxidative stress.", "Our results rather suggest that glycyrrhizin interferes with H5N1-induced oxidative stress. Influenza A virus (including H5N1) infection induces ROS formation. Antioxidants were found to inhibit influenza A virus replication and influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] .", "Antioxidants were found to inhibit influenza A virus replication and influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] . Here, glycyrrhizin interfered with H5N1-induced activation of NFkB, p38, and JNK representing redox-sensitive signalling events [48] [49] [50] [51] involved in influenza A virus replication and influenza A virusinduced cellular cytokine/chemokine production [34, [43] [44] [45] [46] 55] . Glycyrrhizin 50 mg/ml significantly reduced H5N1-induced activation of NFkB.", "Glycyrrhizin 50 mg/ml significantly reduced H5N1-induced activation of NFkB. In addition, glycyrrhizin concentrations as low as 25 mg/ml effectively interfered with H5N1-induced ROS formation and with phosphorylation of the redox-sensitive MAPKs p38 and JNK. In our model, activation of p38 appears to be critical for H5N1-associated redox signalling since p38 inhibition had been shown before to mimick effects of the antioxidant N-acetyl-cysteine (NAC) [34] .", "In our model, activation of p38 appears to be critical for H5N1-associated redox signalling since p38 inhibition had been shown before to mimick effects of the antioxidant N-acetyl-cysteine (NAC) [34] . Interestingly and in contrast to glycyrrhizin, NAC failed to inhibit H5N1 replication or H5N1-induced cytokine/chemokine expression in therapeutically relevant concentrations. Glycyrrhizin diminished H5N1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with H5N1 replication although redox-sensitive signalling pathways have been described to be involved in both processes.", "Glycyrrhizin diminished H5N1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with H5N1 replication although redox-sensitive signalling pathways have been described to be involved in both processes. Therefore, H5N1-induced proinflammatory gene expression appears to be more sensitive to inhibition of ROS formation than H5N1 replication. Indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] .", "Indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] . In a previous report, we could show that similar glycyrrhizin concentrations like those investigated here interfered with H5N1-induced pro-inflammatory gene expression but not with H5N1 replication in human monocyte-derived macrophages [57] . In addition, other immunomodulatory treatment regimens that did not influence H5N1 replication reduced mortality in H5N1-infected mice [31, 58] .", "In addition, other immunomodulatory treatment regimens that did not influence H5N1 replication reduced mortality in H5N1-infected mice [31, 58] . Therefore, glycyrrhizin represents a potential additional treatment option that interfers with both H5N1 replication and H5N1induced expression of pro-inflammatory cytokines in lung cells. Interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8 + T-lymphocytes.", "Interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8 + T-lymphocytes. Global immunosuppressants like corticosteroids failed to protect from lethal influenza virus infection [59] . Moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper NK cell cytolytic activity [60] .", "Moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper NK cell cytolytic activity [60] . In this context, glycyrrhizin had already been shown not to affect natural killer cell activity in the concentrations used here [57] . In conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation SNMC) interfere with highly pathogenic H5N1 influenza A virus replication and H5N1-induced proinflammatory gene expression at least in part through interference with H5N1-induced ROS formation and in turn reduced activation of p38, JNK, and NFkB in lung cells.", "In conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation SNMC) interfere with highly pathogenic H5N1 influenza A virus replication and H5N1-induced proinflammatory gene expression at least in part through interference with H5N1-induced ROS formation and in turn reduced activation of p38, JNK, and NFkB in lung cells. Since we used the clinical formulation SNMC effects of other ingredients like glycin or cystein cannot be excluded. Vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza A virus pandemic [61] .", "Vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza A virus pandemic [61] . Anti-inflammatory and immunomodulatory agents are considered to be important candidates as constituents of anti-influenza treatment strategies that may save lives in an influenza pandemic situation [61] . Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1-caused disease." ]

[ "Glycyrrhizin is known to exert antiviral and anti-inflammatory effects. Here, the effects of an approved parenteral glycyrrhizin preparation (Stronger Neo-Minophafen C) were investigated on highly pathogenic influenza A H5N1 virus replication, H5N1-induced apoptosis, and H5N1-induced pro-inflammatory responses in lung epithelial (A549) cells. Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher).", "Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher). Glycyrrhizin also diminished monocyte migration towards supernatants of H5N1-infected A549 cells. The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen species and (in turn) reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication.", "The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen species and (in turn) reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication. Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease. Text: Highly pathogenic H5N1 influenza A viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] .", "Text: Highly pathogenic H5N1 influenza A viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] . At least for the first wave of an H5N1 pandemic, no sufficient amounts of adequate vaccines will be available [1] [2] [3] [4] [6] [7] [8] . Therefore, antiviral therapy for influenza A viruses including highly pathogenic H5N1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] .", "Therefore, antiviral therapy for influenza A viruses including highly pathogenic H5N1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] . The neuraminidase inhibitors oseltamivir and zanamivir as well as the adamantanes amantadin and rimantadin that interfere with the influenza M2 protein are licensed for the treament of influenza [1] [2] [3] [4] 6] . However, the use of both drug classes is limited by the emergence of resistant virus strains.", "However, the use of both drug classes is limited by the emergence of resistant virus strains. In seasonal influenza strains, the majority of H3N2 viruses and a great proportion of H1N1 viruses in humans are now considered to be amantadine-and rimantadine-resistant [10] [11] [12] [13] . Moreover, a drastic increase in oseltamivir-resistant H1N1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] .", "Moreover, a drastic increase in oseltamivir-resistant H1N1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] . Preliminary data from the United States predict a further rise for the 2008/2009 season, possibly resulting in more than 90% of the circulating H1N1 strains to be oseltamivir resistant [14] . H5N1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza A virus strains and treatment-resistant H5N1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] .", "H5N1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza A virus strains and treatment-resistant H5N1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] . More-over, parenteral agents for the treatment of seriously ill patients are missing. Glycyrrhizin, a triterpene saponine, is a constituent of licorice root.", "Glycyrrhizin, a triterpene saponine, is a constituent of licorice root. It has been found to interfere with replication and/or cytopathogenic effect (CPE) induction of many viruses including respiratory viruses such as respiratory syncytial virus, SARS coronavirus, HIV, and influenza viruses [22] [23] [24] [25] [26] [27] [28] . Moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] .", "Moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] . The severity of human H5N1 disease has been associated with hypercytokinaemia (''cytokine storm'') [29, 30] . Delayed antiviral plus immunomodulator treatment reduced H5N1-induced mortality in mice [31] .", "Delayed antiviral plus immunomodulator treatment reduced H5N1-induced mortality in mice [31] . Therefore, antiinflammatory and immunomodulatory effects exerted by glycyrrhizin may be beneficial for treatment of H5N1. Also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza A virus replication and virus-induced pro-inflammatory responses [32] [33] [34] .", "Also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza A virus replication and virus-induced pro-inflammatory responses [32] [33] [34] . Stronger Neo-Minophagen C (SNMC) is a glycyrrhizin preparation (available as tablets or parenteral formulation) that is approved in Japan for the treatment of chronic hepatic diseases and is marketed in Japan, China, Korea, Taiwan, Indonesia, India, and Mongolia. Here, we investigated the influence of SNMC on H5N1 replication, on H5N1-induced cytokine expression, on H5N1-induced cellular oxidative stress, and on critical H5N1-induced cellular signalling events in human pneumocytes (A549 cell line).", "Here, we investigated the influence of SNMC on H5N1 replication, on H5N1-induced cytokine expression, on H5N1-induced cellular oxidative stress, and on critical H5N1-induced cellular signalling events in human pneumocytes (A549 cell line). Glycyrrhizin (Stronger Neo Minophagen C) was obtained from Minophagen Pharmaceuticals Co., Ltd. (Tokyo, Japan). The influenza strain A/Vietnam/1203/04 (H5N1) was received from the WHO Influenza Centre (National Institute for Medical Research, London, UK).", "The influenza strain A/Vietnam/1203/04 (H5N1) was received from the WHO Influenza Centre (National Institute for Medical Research, London, UK). The H5N1 influenza strain A/Thailand/ 1(Kan-1)/04 was obtained from Prof. Pilaipan Puthavathana (Mahidol University, Bangkok, Thailand). Virus stocks were prepared by infecting Vero cells (African green monkey kidney; ATCC, Manassas, VA) and aliquots were stored at 280uC.", "Virus stocks were prepared by infecting Vero cells (African green monkey kidney; ATCC, Manassas, VA) and aliquots were stored at 280uC. Virus titres were determined as 50% tissue culture infectious dose (TCID 50 /ml) in confluent Vero cells in 96-well microtiter plates. A549 cells (human lung carcinoma; ATCC: CCL-185, obtained from LGC Standards GmbH, Wesel, Germany) were grown at 37uC in minimal essential medium (MEM) supplemented with 10% FBS, 100 IU/ml of penicillin and 100 mg/ml streptomycin.", "A549 cells (human lung carcinoma; ATCC: CCL-185, obtained from LGC Standards GmbH, Wesel, Germany) were grown at 37uC in minimal essential medium (MEM) supplemented with 10% FBS, 100 IU/ml of penicillin and 100 mg/ml streptomycin. Human monocytes were isolated from buffy coats of healthy donors, obtained from Institute of Transfusion Medicine and Immune Haematology, German Red Cross Blood Donor Center, Johann Wolfgang Goethe-University, Frankfurt am Main. After centrifugation on Ficoll (Biocoll)-Hypaque density gradient (Biochrom AG, Berlin, Germany), mononuclear cells were collected from the interface and washed with PBS.", "After centrifugation on Ficoll (Biocoll)-Hypaque density gradient (Biochrom AG, Berlin, Germany), mononuclear cells were collected from the interface and washed with PBS. Then, monocytes were isolated using magnetically labeled CD14 MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) following the manufacturer's instructions. Monocytes were cultivated in IMDM supplemented with 10% pooled human serum, 100 IU/ml of penicillin, and 100 mg/ml streptomycin.", "Monocytes were cultivated in IMDM supplemented with 10% pooled human serum, 100 IU/ml of penicillin, and 100 mg/ml streptomycin. The cellular viability was assessed on confluent cell layers with CellTiter-GloH Luminescent Cell Viability Assay (Promega GmbH, Mannheim, Germany) according to the manufacturers' protocol. Cell viability was expressed as percentage of non-treated control.", "Cell viability was expressed as percentage of non-treated control. To determine intracellular NP localisation, H5N1-infected A549 were fixed 8 hours p.i. for 15 min with ice-cold acetone/ methanol (40:60, Mallinckrodt Baker B.V., Deventer, The Netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in PBS) directed against the influenza A virus nucleoprotein (NP) (Millipore, Molsheim, France).", "for 15 min with ice-cold acetone/ methanol (40:60, Mallinckrodt Baker B.V., Deventer, The Netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in PBS) directed against the influenza A virus nucleoprotein (NP) (Millipore, Molsheim, France). An Alexa Fluor 488 goat anti-mouse IgG (H&L) (Invitrogen, Eugene, Oregon, USA) was used (1 h incubation, 1:1000 in PBS) as secondary antibody. Nuclei were stained using 49,6-diamidino-2phenylindole (DAPI) (Sigma-Aldrich Chemie GmbH, Munich, Germany).", "Nuclei were stained using 49,6-diamidino-2phenylindole (DAPI) (Sigma-Aldrich Chemie GmbH, Munich, Germany). Fluorescence was visualised using Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany). For flow cytometric analysis, the same antibodies were used. The cytopathogenic effect (CPE) reduction assay was performed as described before [34] .", "The cytopathogenic effect (CPE) reduction assay was performed as described before [34] . Confluent A549 cell monolayers grown in 96-well microtitre plates were infected with influenza A strains at the indicated multiplicities of infection (MOIs). After a one hour adsorption period, cells were washed to remove non-detached virus.", "After a one hour adsorption period, cells were washed to remove non-detached virus. The virus-induced CPE was recorded at 24 h post infection (p.i.). Unless otherwise stated, A549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period.", "Unless otherwise stated, A549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period. For time-ofaddition experiments, glycyrrhizin was added exclusively during the 1 h pre-incubation period, exclusively during the 1 h adsorption period, or after exclusively after the wash-out of input virus. Total RNA was isolated from cell cultures using TRI reagent (Sigma-Aldrich, Munich, Germany).", "Total RNA was isolated from cell cultures using TRI reagent (Sigma-Aldrich, Munich, Germany). Real time PCR for H5 was performed using described methods [35] . The following primers were used: sense 59 acg tat gac tac ccg cag tat tca g 39; antisense 59 aga cca gcy acc atg att gc 39; probe 6-FAM-tca aca gtg gcg agt tcc cta gca-TAMRA.", "The following primers were used: sense 59 acg tat gac tac ccg cag tat tca g 39; antisense 59 aga cca gcy acc atg att gc 39; probe 6-FAM-tca aca gtg gcg agt tcc cta gca-TAMRA. The fraction of cells with fractional DNA content (''sub-G1'' cell subpopulation) indicates cytotoxicity. Sub-G1 cells are considered to be dead (usually apoptotic) cells.", "Sub-G1 cells are considered to be dead (usually apoptotic) cells. Cells were fixed with 70% ethanol for two hours at 220uC. The cellular DNA was stained using propidium iodide (20 mg/ml) and analysed by flow cytometry (FacsCalibur, BD Biosciences, Heidelberg, Germany).", "The cellular DNA was stained using propidium iodide (20 mg/ml) and analysed by flow cytometry (FacsCalibur, BD Biosciences, Heidelberg, Germany). Caspase activation was measured using the Caspase-Glo 8, 9, or 3/7 Assays (Promega, Mannheim, Germany) following the manufacturer's instructions. Cell culture supernatants were collected and frozen at 280uC.", "Cell culture supernatants were collected and frozen at 280uC. Cytokines/chemokines were quantified by specific ELISA Duo Sets (R&D Systems GmbH, Wiesbaden, Germany) following the manufacturer's instructions. NFkB activity was investigated in H5N1 (MOI 0.01)-infected cells by quantification of the NFkB subunits Rel A (p65) and NFkB1 (p50) from nuclear extracts using the TransAM TM transcription factor DNA-binding ELISAs (Active Motif, Rixensart, Belgium).", "NFkB activity was investigated in H5N1 (MOI 0.01)-infected cells by quantification of the NFkB subunits Rel A (p65) and NFkB1 (p50) from nuclear extracts using the TransAM TM transcription factor DNA-binding ELISAs (Active Motif, Rixensart, Belgium). Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction. Cell culture supernatants were investigated for chemotactic activity by measurement of the activity to induce monocyte migration through membrane inserts in 24-well plates (pore size 8 mm; BD Biosciences, Heidelberg, Germany).", "Cell culture supernatants were investigated for chemotactic activity by measurement of the activity to induce monocyte migration through membrane inserts in 24-well plates (pore size 8 mm; BD Biosciences, Heidelberg, Germany). Monocytes (1610 6 in 100 ml of IMDM with 10% pooled human serum) were added into the cell culture inserts (upper chamber) and cell culture supernatants (300 ml), were added to the lower chamber of the well. After a 48 h incubation period, cells were fixed with 4% paraformaldehyde and permeabilised with PBS containing 0.3% Tritron X-100.", "After a 48 h incubation period, cells were fixed with 4% paraformaldehyde and permeabilised with PBS containing 0.3% Tritron X-100. Then, nuclei were stained with 49,6-diamidino-2phenylindole (DAPI). The upper side of the membrane was wiped with a wet swab to remove the cells, while the lower side of the membrane was rinsed with PBS.", "The upper side of the membrane was wiped with a wet swab to remove the cells, while the lower side of the membrane was rinsed with PBS. The number of cells at the lower side of each membrane was quantified by counting of cells from three randomly chosen sections (3.7 mm 2 ) using an Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany). Cells were lysed in Triton X-sample buffer and separated by SDS-PAGE.", "Cells were lysed in Triton X-sample buffer and separated by SDS-PAGE. Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction. Proteins were detected using specific antibodies against bactin (Sigma-Aldrich Chemie GmbH, Munich, Germany), JNK, phosphorylated JNK, p38, or phosphorylated p38, (all purchased from New England Biolabs GmbH, Frankfurt am Main, Germany) and were visualised by enhanced chemiluminescence using a commercially available kit (Amersham, Freiburg, Germany).", "Proteins were detected using specific antibodies against bactin (Sigma-Aldrich Chemie GmbH, Munich, Germany), JNK, phosphorylated JNK, p38, or phosphorylated p38, (all purchased from New England Biolabs GmbH, Frankfurt am Main, Germany) and were visualised by enhanced chemiluminescence using a commercially available kit (Amersham, Freiburg, Germany). Reactive oxygen species (ROS) were detected using the Image-iT LIVE Green Reactive Oxygen Species Kit (Molecular Probes, distributed by Invitrogen, Karlsruhe, Germany). Two groups were compared by t-test.", "Two groups were compared by t-test. More groups were compared by ANOVA with subsequent Student-Newman-Keuls test. The A549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type II pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g.", "The A549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type II pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g. 6,37,38]. If not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period.", "If not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period. Glycyrrhizin 200 mg/ml (the maximum tested concentration) did not affect A549 cell viability (data not shown) but clearly decreased CPE formation in A549 cells infected with the H5N1 influenza strain A/Thailand/1(Kan-1)/04 at MOIs of 0.01, 0.1 or 1 ( Figure 1A ). Similar results were obtained in A549 cells infected with strain A/Vietnam/1203/04 (H5N1) (Suppl.", "Similar results were obtained in A549 cells infected with strain A/Vietnam/1203/04 (H5N1) (Suppl. Figure 1A) . Staining of A549 cells for influenza A nucleoprotein 24 h after infection with strain H5N1 A/Thailand/1(Kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza A nucleoprotein positive cells ( Figure 1B) .", "Staining of A549 cells for influenza A nucleoprotein 24 h after infection with strain H5N1 A/Thailand/1(Kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza A nucleoprotein positive cells ( Figure 1B) . To examine the influence of glycyrrhizin on virus progeny, A549 cells were infected with the H5N1 influenza strain A/ Thailand/1(Kan-1)/04 at MOI 0.01 or MOI 1 and infectious virus titres were determined 24 h post infection ( Figure 1C ). While glycyrrhizin in concentrations up to 50 mg/ml did not affect H5N1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (MOI 0.01: 13-fold reduction, MOI 1: 10-fold reduction).", "While glycyrrhizin in concentrations up to 50 mg/ml did not affect H5N1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (MOI 0.01: 13-fold reduction, MOI 1: 10-fold reduction). Next, influence of glycyrrhizin on H5N1 replication was confirmed by the detection of viral (H5) RNA using quantitative PCR. Only glycyrrhizin concentrations $100 mg/ml significantly reduced Figure 1B) or H5N1 A/Vietnam/1203/04-infected (Suppl.", "Only glycyrrhizin concentrations $100 mg/ml significantly reduced Figure 1B) or H5N1 A/Vietnam/1203/04-infected (Suppl. Figure 1C ) A549 cells (MOI 0.01) 24 h post infection. Time-of-addition experiments revealed that maximal effects were achieved when glycyrrhizin was continuously present starting with a 1 h pre-incubation period ( Figure 1D ).", "Time-of-addition experiments revealed that maximal effects were achieved when glycyrrhizin was continuously present starting with a 1 h pre-incubation period ( Figure 1D ). Addition of glycyrrhizin post infection showed reduced antiviral effects while pre-incubation alone or glycyrrhizin addition during the adsorption period did not significantly affect H5N1 replication. For investigation of H5N1-induced cytokine expression, five pro-inflammatory genes were chosen that had been correlated to severity of influenza disease: CXCL10 (also known as interferon-cinducible protein 10, IP-10), interleukin 6 (IL6), interleukin 8, (IL8; also known as CXCL8), CCL2 (also known as monocyte chemoattractant protein 1, MCP-1), and CCL5 (also known as RANTES).", "For investigation of H5N1-induced cytokine expression, five pro-inflammatory genes were chosen that had been correlated to severity of influenza disease: CXCL10 (also known as interferon-cinducible protein 10, IP-10), interleukin 6 (IL6), interleukin 8, (IL8; also known as CXCL8), CCL2 (also known as monocyte chemoattractant protein 1, MCP-1), and CCL5 (also known as RANTES). A549 cells were infected with H5N1 A/Thailand/ 1(Kan-1)/04 or H5N1 A/Vietnam/1203/04 at MOI 0.01, 0.1, or 1. Glycyrrhizin treatment was performed with 25, 50, 100, or 200 mg/ml.", "Glycyrrhizin treatment was performed with 25, 50, 100, or 200 mg/ml. Cytokine expression was detected 24 h post infection by ELISA. Glycyrrhizin did not affect cytokine expression of noninfected cells (data not shown) but inhibited expression of all cytokines investigated in H5N1-infected cells in a dose-dependent manner (Figure 2, Figure 3A ).", "Glycyrrhizin did not affect cytokine expression of noninfected cells (data not shown) but inhibited expression of all cytokines investigated in H5N1-infected cells in a dose-dependent manner (Figure 2, Figure 3A ). Effects were more pronounced at lower MOIs. Notably, expression of all cytokines except IL8 was significantly inhibited after treatment with glycyrrhizin 50 mg/ml Figure 3A ) although these glycyrrhizin concentrations had no effect on H5N1 replication in A549 cells (Figure 1, Figure S1 ).", "Notably, expression of all cytokines except IL8 was significantly inhibited after treatment with glycyrrhizin 50 mg/ml Figure 3A ) although these glycyrrhizin concentrations had no effect on H5N1 replication in A549 cells (Figure 1, Figure S1 ). Cytokine expression by influenza A virus-infected respiratory cells causes recruitment of peripheral blood monocytes into the lungs of patients where they differentiate to macrophages which are thought to contribute to influenza A virus pathogenicity [5, 39] . In a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.1)-infected A549 cells through 8 mm filters.", "In a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.1)-infected A549 cells through 8 mm filters. Monocyte migration towards supernatants of H5N1-infected cells was strongly increased relative to migration towards supernatants of non-infected cells. Treatment of H5N1- infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( Figure 3B ).", "Treatment of H5N1- infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( Figure 3B ). Influenza viruses including H5N1 have been shown to induce caspase-dependent apoptosis in airway cells and this apoptosis has been correlated to the virus pathogenicity [40, 41] . Glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( Figure 4A-C) .", "Glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( Figure 4A-C) . Glycyrrhizin concentrations $100 mg/ml inhibited H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.01)-induced activation of the initiator caspases 8 and 9 as well as of the effector caspases 3/7 in A549 cells as determined 24 h post infection ( Figure 4A-C) . Lower glycyrrhizin concentrations did not affect H5N1-induced apoptosis.", "Lower glycyrrhizin concentrations did not affect H5N1-induced apoptosis. The detection of cells in sub-G1 phase resulted in similar findings ( Figure 4D ). Substances that inhibit H5N1-induced caspase 3 activation including caspase 3 inhibitors cause nuclear retention of RNP complexes [34, 42] .", "Substances that inhibit H5N1-induced caspase 3 activation including caspase 3 inhibitors cause nuclear retention of RNP complexes [34, 42] . In accordance, glycyrrhizin also interfered with nuclear export RNP at MOI 1 ( Figure S2 ). Similar results were obtained in MOI 0.01 H5N1 A/Thailand/1(Kan-1)/04infected cells ( Figure S3 ).", "Similar results were obtained in MOI 0.01 H5N1 A/Thailand/1(Kan-1)/04infected cells ( Figure S3 ). Influence of glycyrrhizin on H5N1-induced activation of nuclear factor kB (NFkB), p38, and on H5N1-induced cellular reactive oxygen species (ROS) formation Activation of NFkB, p38, and JNK have been associated with influenza A virus replication and virus-induced pro-inflammatory gene expression [34, [43] [44] [45] [46] [47] . While glycyrrhizin did not influence NFkB activity in non-infected A549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited NFkB activation in H5N1-infected cells ( Figure 5A ).", "While glycyrrhizin did not influence NFkB activity in non-infected A549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited NFkB activation in H5N1-infected cells ( Figure 5A ). Moreover, glycyrrhizin inhibited H5N1-induced phosphorylation of the MAPKs p38 and JNK ( Figure 5B ). In addition to their roles during influenza A virus replication and virus-induced cytokine/chemokine expression, NFkB, p38, and JNK are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] .", "In addition to their roles during influenza A virus replication and virus-induced cytokine/chemokine expression, NFkB, p38, and JNK are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] . Antioxidants had been already found to interfere with influenza A virus-induced signalling through NFkB, p38, and JNK, with influenza A virus replication, and with influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] . Since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with H5N1-induced ROS formation.", "Since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with H5N1-induced ROS formation. Indeed glycyrrhizin exerted clear antioxidative effects in H5N1 (MOI 0.01)-infected cells ( Figure 5C ) causing significant reduction of ROS formation already at a concentration of 25 mg/ml ( Figure 5D ). Here, we show that glycyrrhizin inhibits the replication of highly pathogenic H5N1 influenza A virus, H5N1-induced apoptosis, and H5N1-induced expression of pro-inflammatory cytokines in lung-derived A549 cells.", "Here, we show that glycyrrhizin inhibits the replication of highly pathogenic H5N1 influenza A virus, H5N1-induced apoptosis, and H5N1-induced expression of pro-inflammatory cytokines in lung-derived A549 cells. After intravenous administration, achievable plasma concentrations of glycyrrhizin have been described to be about 100 mg/ml [52] . Therefore, the glycyrrhizin concentrations found to interfere with H5N1 replication and H5N1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels.", "Therefore, the glycyrrhizin concentrations found to interfere with H5N1 replication and H5N1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels. Notably, although higher glycyrrhizin concentrations were needed to interfere with SARS coronavirus replication [22] than with H5N1 replication, beneficial results were reported in glycyrrhizin (SNMC)-treated SARS patients in comparison to SARS patients who did not receive glycyrrhizin [23] . Notably, investigation of different glycyrrhizin derivatives against SARS coronavirus led to the identification of compounds with enhanced antiviral activity [53] .", "Notably, investigation of different glycyrrhizin derivatives against SARS coronavirus led to the identification of compounds with enhanced antiviral activity [53] . Therefore, glycyrrhizin might also serve as lead structure for the development of novel anti-influenza drugs. Experimental results suggested that glycyrrhizin might be able to affect seasonal influenza A virus disease by antiviral and immunomodulatory effects [26, 27] .", "Experimental results suggested that glycyrrhizin might be able to affect seasonal influenza A virus disease by antiviral and immunomodulatory effects [26, 27] . Mice were prevented from lethal H2N2 infection by glycyrrhizin although no influence on virus replication was detected. The mechanism was suggested to be induction of interferon-c in T-cells by glycyrrhizin [54] .", "The mechanism was suggested to be induction of interferon-c in T-cells by glycyrrhizin [54] . Moreover, glycyrrhizin was shown to influence seasonal influenza A virus replication through interaction with the cell membrane [25, 28] . However, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period.", "However, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period. Since glycyrrhizin addition during the adsorption period did not influence H5N1 replication in our experiments it appears not likely that membrane effects contribute to anti-H5N1 effects detected here in lower concentrations. Our results rather suggest that glycyrrhizin interferes with H5N1-induced oxidative stress.", "Our results rather suggest that glycyrrhizin interferes with H5N1-induced oxidative stress. Influenza A virus (including H5N1) infection induces ROS formation. Antioxidants were found to inhibit influenza A virus replication and influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] .", "Antioxidants were found to inhibit influenza A virus replication and influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] . Here, glycyrrhizin interfered with H5N1-induced activation of NFkB, p38, and JNK representing redox-sensitive signalling events [48] [49] [50] [51] involved in influenza A virus replication and influenza A virusinduced cellular cytokine/chemokine production [34, [43] [44] [45] [46] 55] . Glycyrrhizin 50 mg/ml significantly reduced H5N1-induced activation of NFkB.", "Glycyrrhizin 50 mg/ml significantly reduced H5N1-induced activation of NFkB. In addition, glycyrrhizin concentrations as low as 25 mg/ml effectively interfered with H5N1-induced ROS formation and with phosphorylation of the redox-sensitive MAPKs p38 and JNK. In our model, activation of p38 appears to be critical for H5N1-associated redox signalling since p38 inhibition had been shown before to mimick effects of the antioxidant N-acetyl-cysteine (NAC) [34] .", "In our model, activation of p38 appears to be critical for H5N1-associated redox signalling since p38 inhibition had been shown before to mimick effects of the antioxidant N-acetyl-cysteine (NAC) [34] . Interestingly and in contrast to glycyrrhizin, NAC failed to inhibit H5N1 replication or H5N1-induced cytokine/chemokine expression in therapeutically relevant concentrations. Glycyrrhizin diminished H5N1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with H5N1 replication although redox-sensitive signalling pathways have been described to be involved in both processes.", "Glycyrrhizin diminished H5N1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with H5N1 replication although redox-sensitive signalling pathways have been described to be involved in both processes. Therefore, H5N1-induced proinflammatory gene expression appears to be more sensitive to inhibition of ROS formation than H5N1 replication. Indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] .", "Indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] . In a previous report, we could show that similar glycyrrhizin concentrations like those investigated here interfered with H5N1-induced pro-inflammatory gene expression but not with H5N1 replication in human monocyte-derived macrophages [57] . In addition, other immunomodulatory treatment regimens that did not influence H5N1 replication reduced mortality in H5N1-infected mice [31, 58] .", "In addition, other immunomodulatory treatment regimens that did not influence H5N1 replication reduced mortality in H5N1-infected mice [31, 58] . Therefore, glycyrrhizin represents a potential additional treatment option that interfers with both H5N1 replication and H5N1induced expression of pro-inflammatory cytokines in lung cells. Interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8 + T-lymphocytes.", "Interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8 + T-lymphocytes. Global immunosuppressants like corticosteroids failed to protect from lethal influenza virus infection [59] . Moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper NK cell cytolytic activity [60] .", "Moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper NK cell cytolytic activity [60] . In this context, glycyrrhizin had already been shown not to affect natural killer cell activity in the concentrations used here [57] . In conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation SNMC) interfere with highly pathogenic H5N1 influenza A virus replication and H5N1-induced proinflammatory gene expression at least in part through interference with H5N1-induced ROS formation and in turn reduced activation of p38, JNK, and NFkB in lung cells.", "In conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation SNMC) interfere with highly pathogenic H5N1 influenza A virus replication and H5N1-induced proinflammatory gene expression at least in part through interference with H5N1-induced ROS formation and in turn reduced activation of p38, JNK, and NFkB in lung cells. Since we used the clinical formulation SNMC effects of other ingredients like glycin or cystein cannot be excluded. Vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza A virus pandemic [61] .", "Vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza A virus pandemic [61] . Anti-inflammatory and immunomodulatory agents are considered to be important candidates as constituents of anti-influenza treatment strategies that may save lives in an influenza pandemic situation [61] . Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1-caused disease." ]

[ "Glycyrrhizin is known to exert antiviral and anti-inflammatory effects. Here, the effects of an approved parenteral glycyrrhizin preparation (Stronger Neo-Minophafen C) were investigated on highly pathogenic influenza A H5N1 virus replication, H5N1-induced apoptosis, and H5N1-induced pro-inflammatory responses in lung epithelial (A549) cells. Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher).", "Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher). Glycyrrhizin also diminished monocyte migration towards supernatants of H5N1-infected A549 cells. The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen species and (in turn) reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication.", "The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen species and (in turn) reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication. Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease. Text: Highly pathogenic H5N1 influenza A viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] .", "Text: Highly pathogenic H5N1 influenza A viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] . At least for the first wave of an H5N1 pandemic, no sufficient amounts of adequate vaccines will be available [1] [2] [3] [4] [6] [7] [8] . Therefore, antiviral therapy for influenza A viruses including highly pathogenic H5N1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] .", "Therefore, antiviral therapy for influenza A viruses including highly pathogenic H5N1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] . The neuraminidase inhibitors oseltamivir and zanamivir as well as the adamantanes amantadin and rimantadin that interfere with the influenza M2 protein are licensed for the treament of influenza [1] [2] [3] [4] 6] . However, the use of both drug classes is limited by the emergence of resistant virus strains.", "However, the use of both drug classes is limited by the emergence of resistant virus strains. In seasonal influenza strains, the majority of H3N2 viruses and a great proportion of H1N1 viruses in humans are now considered to be amantadine-and rimantadine-resistant [10] [11] [12] [13] . Moreover, a drastic increase in oseltamivir-resistant H1N1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] .", "Moreover, a drastic increase in oseltamivir-resistant H1N1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] . Preliminary data from the United States predict a further rise for the 2008/2009 season, possibly resulting in more than 90% of the circulating H1N1 strains to be oseltamivir resistant [14] . H5N1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza A virus strains and treatment-resistant H5N1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] .", "H5N1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza A virus strains and treatment-resistant H5N1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] . More-over, parenteral agents for the treatment of seriously ill patients are missing. Glycyrrhizin, a triterpene saponine, is a constituent of licorice root.", "Glycyrrhizin, a triterpene saponine, is a constituent of licorice root. It has been found to interfere with replication and/or cytopathogenic effect (CPE) induction of many viruses including respiratory viruses such as respiratory syncytial virus, SARS coronavirus, HIV, and influenza viruses [22] [23] [24] [25] [26] [27] [28] . Moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] .", "Moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] . The severity of human H5N1 disease has been associated with hypercytokinaemia (''cytokine storm'') [29, 30] . Delayed antiviral plus immunomodulator treatment reduced H5N1-induced mortality in mice [31] .", "Delayed antiviral plus immunomodulator treatment reduced H5N1-induced mortality in mice [31] . Therefore, antiinflammatory and immunomodulatory effects exerted by glycyrrhizin may be beneficial for treatment of H5N1. Also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza A virus replication and virus-induced pro-inflammatory responses [32] [33] [34] .", "Also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza A virus replication and virus-induced pro-inflammatory responses [32] [33] [34] . Stronger Neo-Minophagen C (SNMC) is a glycyrrhizin preparation (available as tablets or parenteral formulation) that is approved in Japan for the treatment of chronic hepatic diseases and is marketed in Japan, China, Korea, Taiwan, Indonesia, India, and Mongolia. Here, we investigated the influence of SNMC on H5N1 replication, on H5N1-induced cytokine expression, on H5N1-induced cellular oxidative stress, and on critical H5N1-induced cellular signalling events in human pneumocytes (A549 cell line).", "Here, we investigated the influence of SNMC on H5N1 replication, on H5N1-induced cytokine expression, on H5N1-induced cellular oxidative stress, and on critical H5N1-induced cellular signalling events in human pneumocytes (A549 cell line). Glycyrrhizin (Stronger Neo Minophagen C) was obtained from Minophagen Pharmaceuticals Co., Ltd. (Tokyo, Japan). The influenza strain A/Vietnam/1203/04 (H5N1) was received from the WHO Influenza Centre (National Institute for Medical Research, London, UK).", "The influenza strain A/Vietnam/1203/04 (H5N1) was received from the WHO Influenza Centre (National Institute for Medical Research, London, UK). The H5N1 influenza strain A/Thailand/ 1(Kan-1)/04 was obtained from Prof. Pilaipan Puthavathana (Mahidol University, Bangkok, Thailand). Virus stocks were prepared by infecting Vero cells (African green monkey kidney; ATCC, Manassas, VA) and aliquots were stored at 280uC.", "Virus stocks were prepared by infecting Vero cells (African green monkey kidney; ATCC, Manassas, VA) and aliquots were stored at 280uC. Virus titres were determined as 50% tissue culture infectious dose (TCID 50 /ml) in confluent Vero cells in 96-well microtiter plates. A549 cells (human lung carcinoma; ATCC: CCL-185, obtained from LGC Standards GmbH, Wesel, Germany) were grown at 37uC in minimal essential medium (MEM) supplemented with 10% FBS, 100 IU/ml of penicillin and 100 mg/ml streptomycin.", "A549 cells (human lung carcinoma; ATCC: CCL-185, obtained from LGC Standards GmbH, Wesel, Germany) were grown at 37uC in minimal essential medium (MEM) supplemented with 10% FBS, 100 IU/ml of penicillin and 100 mg/ml streptomycin. Human monocytes were isolated from buffy coats of healthy donors, obtained from Institute of Transfusion Medicine and Immune Haematology, German Red Cross Blood Donor Center, Johann Wolfgang Goethe-University, Frankfurt am Main. After centrifugation on Ficoll (Biocoll)-Hypaque density gradient (Biochrom AG, Berlin, Germany), mononuclear cells were collected from the interface and washed with PBS.", "After centrifugation on Ficoll (Biocoll)-Hypaque density gradient (Biochrom AG, Berlin, Germany), mononuclear cells were collected from the interface and washed with PBS. Then, monocytes were isolated using magnetically labeled CD14 MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) following the manufacturer's instructions. Monocytes were cultivated in IMDM supplemented with 10% pooled human serum, 100 IU/ml of penicillin, and 100 mg/ml streptomycin.", "Monocytes were cultivated in IMDM supplemented with 10% pooled human serum, 100 IU/ml of penicillin, and 100 mg/ml streptomycin. The cellular viability was assessed on confluent cell layers with CellTiter-GloH Luminescent Cell Viability Assay (Promega GmbH, Mannheim, Germany) according to the manufacturers' protocol. Cell viability was expressed as percentage of non-treated control.", "Cell viability was expressed as percentage of non-treated control. To determine intracellular NP localisation, H5N1-infected A549 were fixed 8 hours p.i. for 15 min with ice-cold acetone/ methanol (40:60, Mallinckrodt Baker B.V., Deventer, The Netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in PBS) directed against the influenza A virus nucleoprotein (NP) (Millipore, Molsheim, France).", "for 15 min with ice-cold acetone/ methanol (40:60, Mallinckrodt Baker B.V., Deventer, The Netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in PBS) directed against the influenza A virus nucleoprotein (NP) (Millipore, Molsheim, France). An Alexa Fluor 488 goat anti-mouse IgG (H&L) (Invitrogen, Eugene, Oregon, USA) was used (1 h incubation, 1:1000 in PBS) as secondary antibody. Nuclei were stained using 49,6-diamidino-2phenylindole (DAPI) (Sigma-Aldrich Chemie GmbH, Munich, Germany).", "Nuclei were stained using 49,6-diamidino-2phenylindole (DAPI) (Sigma-Aldrich Chemie GmbH, Munich, Germany). Fluorescence was visualised using Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany). For flow cytometric analysis, the same antibodies were used. The cytopathogenic effect (CPE) reduction assay was performed as described before [34] .", "The cytopathogenic effect (CPE) reduction assay was performed as described before [34] . Confluent A549 cell monolayers grown in 96-well microtitre plates were infected with influenza A strains at the indicated multiplicities of infection (MOIs). After a one hour adsorption period, cells were washed to remove non-detached virus.", "After a one hour adsorption period, cells were washed to remove non-detached virus. The virus-induced CPE was recorded at 24 h post infection (p.i.). Unless otherwise stated, A549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period.", "Unless otherwise stated, A549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period. For time-ofaddition experiments, glycyrrhizin was added exclusively during the 1 h pre-incubation period, exclusively during the 1 h adsorption period, or after exclusively after the wash-out of input virus. Total RNA was isolated from cell cultures using TRI reagent (Sigma-Aldrich, Munich, Germany).", "Total RNA was isolated from cell cultures using TRI reagent (Sigma-Aldrich, Munich, Germany). Real time PCR for H5 was performed using described methods [35] . The following primers were used: sense 59 acg tat gac tac ccg cag tat tca g 39; antisense 59 aga cca gcy acc atg att gc 39; probe 6-FAM-tca aca gtg gcg agt tcc cta gca-TAMRA.", "The following primers were used: sense 59 acg tat gac tac ccg cag tat tca g 39; antisense 59 aga cca gcy acc atg att gc 39; probe 6-FAM-tca aca gtg gcg agt tcc cta gca-TAMRA. The fraction of cells with fractional DNA content (''sub-G1'' cell subpopulation) indicates cytotoxicity. Sub-G1 cells are considered to be dead (usually apoptotic) cells.", "Sub-G1 cells are considered to be dead (usually apoptotic) cells. Cells were fixed with 70% ethanol for two hours at 220uC. The cellular DNA was stained using propidium iodide (20 mg/ml) and analysed by flow cytometry (FacsCalibur, BD Biosciences, Heidelberg, Germany).", "The cellular DNA was stained using propidium iodide (20 mg/ml) and analysed by flow cytometry (FacsCalibur, BD Biosciences, Heidelberg, Germany). Caspase activation was measured using the Caspase-Glo 8, 9, or 3/7 Assays (Promega, Mannheim, Germany) following the manufacturer's instructions. Cell culture supernatants were collected and frozen at 280uC.", "Cell culture supernatants were collected and frozen at 280uC. Cytokines/chemokines were quantified by specific ELISA Duo Sets (R&D Systems GmbH, Wiesbaden, Germany) following the manufacturer's instructions. NFkB activity was investigated in H5N1 (MOI 0.01)-infected cells by quantification of the NFkB subunits Rel A (p65) and NFkB1 (p50) from nuclear extracts using the TransAM TM transcription factor DNA-binding ELISAs (Active Motif, Rixensart, Belgium).", "NFkB activity was investigated in H5N1 (MOI 0.01)-infected cells by quantification of the NFkB subunits Rel A (p65) and NFkB1 (p50) from nuclear extracts using the TransAM TM transcription factor DNA-binding ELISAs (Active Motif, Rixensart, Belgium). Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction. Cell culture supernatants were investigated for chemotactic activity by measurement of the activity to induce monocyte migration through membrane inserts in 24-well plates (pore size 8 mm; BD Biosciences, Heidelberg, Germany).", "Cell culture supernatants were investigated for chemotactic activity by measurement of the activity to induce monocyte migration through membrane inserts in 24-well plates (pore size 8 mm; BD Biosciences, Heidelberg, Germany). Monocytes (1610 6 in 100 ml of IMDM with 10% pooled human serum) were added into the cell culture inserts (upper chamber) and cell culture supernatants (300 ml), were added to the lower chamber of the well. After a 48 h incubation period, cells were fixed with 4% paraformaldehyde and permeabilised with PBS containing 0.3% Tritron X-100.", "After a 48 h incubation period, cells were fixed with 4% paraformaldehyde and permeabilised with PBS containing 0.3% Tritron X-100. Then, nuclei were stained with 49,6-diamidino-2phenylindole (DAPI). The upper side of the membrane was wiped with a wet swab to remove the cells, while the lower side of the membrane was rinsed with PBS.", "The upper side of the membrane was wiped with a wet swab to remove the cells, while the lower side of the membrane was rinsed with PBS. The number of cells at the lower side of each membrane was quantified by counting of cells from three randomly chosen sections (3.7 mm 2 ) using an Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany). Cells were lysed in Triton X-sample buffer and separated by SDS-PAGE.", "Cells were lysed in Triton X-sample buffer and separated by SDS-PAGE. Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction. Proteins were detected using specific antibodies against bactin (Sigma-Aldrich Chemie GmbH, Munich, Germany), JNK, phosphorylated JNK, p38, or phosphorylated p38, (all purchased from New England Biolabs GmbH, Frankfurt am Main, Germany) and were visualised by enhanced chemiluminescence using a commercially available kit (Amersham, Freiburg, Germany).", "Proteins were detected using specific antibodies against bactin (Sigma-Aldrich Chemie GmbH, Munich, Germany), JNK, phosphorylated JNK, p38, or phosphorylated p38, (all purchased from New England Biolabs GmbH, Frankfurt am Main, Germany) and were visualised by enhanced chemiluminescence using a commercially available kit (Amersham, Freiburg, Germany). Reactive oxygen species (ROS) were detected using the Image-iT LIVE Green Reactive Oxygen Species Kit (Molecular Probes, distributed by Invitrogen, Karlsruhe, Germany). Two groups were compared by t-test.", "Two groups were compared by t-test. More groups were compared by ANOVA with subsequent Student-Newman-Keuls test. The A549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type II pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g.", "The A549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type II pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g. 6,37,38]. If not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period.", "If not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period. Glycyrrhizin 200 mg/ml (the maximum tested concentration) did not affect A549 cell viability (data not shown) but clearly decreased CPE formation in A549 cells infected with the H5N1 influenza strain A/Thailand/1(Kan-1)/04 at MOIs of 0.01, 0.1 or 1 ( Figure 1A ). Similar results were obtained in A549 cells infected with strain A/Vietnam/1203/04 (H5N1) (Suppl.", "Similar results were obtained in A549 cells infected with strain A/Vietnam/1203/04 (H5N1) (Suppl. Figure 1A) . Staining of A549 cells for influenza A nucleoprotein 24 h after infection with strain H5N1 A/Thailand/1(Kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza A nucleoprotein positive cells ( Figure 1B) .", "Staining of A549 cells for influenza A nucleoprotein 24 h after infection with strain H5N1 A/Thailand/1(Kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza A nucleoprotein positive cells ( Figure 1B) . To examine the influence of glycyrrhizin on virus progeny, A549 cells were infected with the H5N1 influenza strain A/ Thailand/1(Kan-1)/04 at MOI 0.01 or MOI 1 and infectious virus titres were determined 24 h post infection ( Figure 1C ). While glycyrrhizin in concentrations up to 50 mg/ml did not affect H5N1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (MOI 0.01: 13-fold reduction, MOI 1: 10-fold reduction).", "While glycyrrhizin in concentrations up to 50 mg/ml did not affect H5N1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (MOI 0.01: 13-fold reduction, MOI 1: 10-fold reduction). Next, influence of glycyrrhizin on H5N1 replication was confirmed by the detection of viral (H5) RNA using quantitative PCR. Only glycyrrhizin concentrations $100 mg/ml significantly reduced Figure 1B) or H5N1 A/Vietnam/1203/04-infected (Suppl.", "Only glycyrrhizin concentrations $100 mg/ml significantly reduced Figure 1B) or H5N1 A/Vietnam/1203/04-infected (Suppl. Figure 1C ) A549 cells (MOI 0.01) 24 h post infection. Time-of-addition experiments revealed that maximal effects were achieved when glycyrrhizin was continuously present starting with a 1 h pre-incubation period ( Figure 1D ).", "Time-of-addition experiments revealed that maximal effects were achieved when glycyrrhizin was continuously present starting with a 1 h pre-incubation period ( Figure 1D ). Addition of glycyrrhizin post infection showed reduced antiviral effects while pre-incubation alone or glycyrrhizin addition during the adsorption period did not significantly affect H5N1 replication. For investigation of H5N1-induced cytokine expression, five pro-inflammatory genes were chosen that had been correlated to severity of influenza disease: CXCL10 (also known as interferon-cinducible protein 10, IP-10), interleukin 6 (IL6), interleukin 8, (IL8; also known as CXCL8), CCL2 (also known as monocyte chemoattractant protein 1, MCP-1), and CCL5 (also known as RANTES).", "For investigation of H5N1-induced cytokine expression, five pro-inflammatory genes were chosen that had been correlated to severity of influenza disease: CXCL10 (also known as interferon-cinducible protein 10, IP-10), interleukin 6 (IL6), interleukin 8, (IL8; also known as CXCL8), CCL2 (also known as monocyte chemoattractant protein 1, MCP-1), and CCL5 (also known as RANTES). A549 cells were infected with H5N1 A/Thailand/ 1(Kan-1)/04 or H5N1 A/Vietnam/1203/04 at MOI 0.01, 0.1, or 1. Glycyrrhizin treatment was performed with 25, 50, 100, or 200 mg/ml.", "Glycyrrhizin treatment was performed with 25, 50, 100, or 200 mg/ml. Cytokine expression was detected 24 h post infection by ELISA. Glycyrrhizin did not affect cytokine expression of noninfected cells (data not shown) but inhibited expression of all cytokines investigated in H5N1-infected cells in a dose-dependent manner (Figure 2, Figure 3A ).", "Glycyrrhizin did not affect cytokine expression of noninfected cells (data not shown) but inhibited expression of all cytokines investigated in H5N1-infected cells in a dose-dependent manner (Figure 2, Figure 3A ). Effects were more pronounced at lower MOIs. Notably, expression of all cytokines except IL8 was significantly inhibited after treatment with glycyrrhizin 50 mg/ml Figure 3A ) although these glycyrrhizin concentrations had no effect on H5N1 replication in A549 cells (Figure 1, Figure S1 ).", "Notably, expression of all cytokines except IL8 was significantly inhibited after treatment with glycyrrhizin 50 mg/ml Figure 3A ) although these glycyrrhizin concentrations had no effect on H5N1 replication in A549 cells (Figure 1, Figure S1 ). Cytokine expression by influenza A virus-infected respiratory cells causes recruitment of peripheral blood monocytes into the lungs of patients where they differentiate to macrophages which are thought to contribute to influenza A virus pathogenicity [5, 39] . In a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.1)-infected A549 cells through 8 mm filters.", "In a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.1)-infected A549 cells through 8 mm filters. Monocyte migration towards supernatants of H5N1-infected cells was strongly increased relative to migration towards supernatants of non-infected cells. Treatment of H5N1- infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( Figure 3B ).", "Treatment of H5N1- infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( Figure 3B ). Influenza viruses including H5N1 have been shown to induce caspase-dependent apoptosis in airway cells and this apoptosis has been correlated to the virus pathogenicity [40, 41] . Glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( Figure 4A-C) .", "Glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( Figure 4A-C) . Glycyrrhizin concentrations $100 mg/ml inhibited H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.01)-induced activation of the initiator caspases 8 and 9 as well as of the effector caspases 3/7 in A549 cells as determined 24 h post infection ( Figure 4A-C) . Lower glycyrrhizin concentrations did not affect H5N1-induced apoptosis.", "Lower glycyrrhizin concentrations did not affect H5N1-induced apoptosis. The detection of cells in sub-G1 phase resulted in similar findings ( Figure 4D ). Substances that inhibit H5N1-induced caspase 3 activation including caspase 3 inhibitors cause nuclear retention of RNP complexes [34, 42] .", "Substances that inhibit H5N1-induced caspase 3 activation including caspase 3 inhibitors cause nuclear retention of RNP complexes [34, 42] . In accordance, glycyrrhizin also interfered with nuclear export RNP at MOI 1 ( Figure S2 ). Similar results were obtained in MOI 0.01 H5N1 A/Thailand/1(Kan-1)/04infected cells ( Figure S3 ).", "Similar results were obtained in MOI 0.01 H5N1 A/Thailand/1(Kan-1)/04infected cells ( Figure S3 ). Influence of glycyrrhizin on H5N1-induced activation of nuclear factor kB (NFkB), p38, and on H5N1-induced cellular reactive oxygen species (ROS) formation Activation of NFkB, p38, and JNK have been associated with influenza A virus replication and virus-induced pro-inflammatory gene expression [34, [43] [44] [45] [46] [47] . While glycyrrhizin did not influence NFkB activity in non-infected A549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited NFkB activation in H5N1-infected cells ( Figure 5A ).", "While glycyrrhizin did not influence NFkB activity in non-infected A549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited NFkB activation in H5N1-infected cells ( Figure 5A ). Moreover, glycyrrhizin inhibited H5N1-induced phosphorylation of the MAPKs p38 and JNK ( Figure 5B ). In addition to their roles during influenza A virus replication and virus-induced cytokine/chemokine expression, NFkB, p38, and JNK are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] .", "In addition to their roles during influenza A virus replication and virus-induced cytokine/chemokine expression, NFkB, p38, and JNK are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] . Antioxidants had been already found to interfere with influenza A virus-induced signalling through NFkB, p38, and JNK, with influenza A virus replication, and with influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] . Since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with H5N1-induced ROS formation.", "Since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with H5N1-induced ROS formation. Indeed glycyrrhizin exerted clear antioxidative effects in H5N1 (MOI 0.01)-infected cells ( Figure 5C ) causing significant reduction of ROS formation already at a concentration of 25 mg/ml ( Figure 5D ). Here, we show that glycyrrhizin inhibits the replication of highly pathogenic H5N1 influenza A virus, H5N1-induced apoptosis, and H5N1-induced expression of pro-inflammatory cytokines in lung-derived A549 cells.", "Here, we show that glycyrrhizin inhibits the replication of highly pathogenic H5N1 influenza A virus, H5N1-induced apoptosis, and H5N1-induced expression of pro-inflammatory cytokines in lung-derived A549 cells. After intravenous administration, achievable plasma concentrations of glycyrrhizin have been described to be about 100 mg/ml [52] . Therefore, the glycyrrhizin concentrations found to interfere with H5N1 replication and H5N1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels.", "Therefore, the glycyrrhizin concentrations found to interfere with H5N1 replication and H5N1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels. Notably, although higher glycyrrhizin concentrations were needed to interfere with SARS coronavirus replication [22] than with H5N1 replication, beneficial results were reported in glycyrrhizin (SNMC)-treated SARS patients in comparison to SARS patients who did not receive glycyrrhizin [23] . Notably, investigation of different glycyrrhizin derivatives against SARS coronavirus led to the identification of compounds with enhanced antiviral activity [53] .", "Notably, investigation of different glycyrrhizin derivatives against SARS coronavirus led to the identification of compounds with enhanced antiviral activity [53] . Therefore, glycyrrhizin might also serve as lead structure for the development of novel anti-influenza drugs. Experimental results suggested that glycyrrhizin might be able to affect seasonal influenza A virus disease by antiviral and immunomodulatory effects [26, 27] .", "Experimental results suggested that glycyrrhizin might be able to affect seasonal influenza A virus disease by antiviral and immunomodulatory effects [26, 27] . Mice were prevented from lethal H2N2 infection by glycyrrhizin although no influence on virus replication was detected. The mechanism was suggested to be induction of interferon-c in T-cells by glycyrrhizin [54] .", "The mechanism was suggested to be induction of interferon-c in T-cells by glycyrrhizin [54] . Moreover, glycyrrhizin was shown to influence seasonal influenza A virus replication through interaction with the cell membrane [25, 28] . However, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period.", "However, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period. Since glycyrrhizin addition during the adsorption period did not influence H5N1 replication in our experiments it appears not likely that membrane effects contribute to anti-H5N1 effects detected here in lower concentrations. Our results rather suggest that glycyrrhizin interferes with H5N1-induced oxidative stress.", "Our results rather suggest that glycyrrhizin interferes with H5N1-induced oxidative stress. Influenza A virus (including H5N1) infection induces ROS formation. Antioxidants were found to inhibit influenza A virus replication and influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] .", "Antioxidants were found to inhibit influenza A virus replication and influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] . Here, glycyrrhizin interfered with H5N1-induced activation of NFkB, p38, and JNK representing redox-sensitive signalling events [48] [49] [50] [51] involved in influenza A virus replication and influenza A virusinduced cellular cytokine/chemokine production [34, [43] [44] [45] [46] 55] . Glycyrrhizin 50 mg/ml significantly reduced H5N1-induced activation of NFkB.", "Glycyrrhizin 50 mg/ml significantly reduced H5N1-induced activation of NFkB. In addition, glycyrrhizin concentrations as low as 25 mg/ml effectively interfered with H5N1-induced ROS formation and with phosphorylation of the redox-sensitive MAPKs p38 and JNK. In our model, activation of p38 appears to be critical for H5N1-associated redox signalling since p38 inhibition had been shown before to mimick effects of the antioxidant N-acetyl-cysteine (NAC) [34] .", "In our model, activation of p38 appears to be critical for H5N1-associated redox signalling since p38 inhibition had been shown before to mimick effects of the antioxidant N-acetyl-cysteine (NAC) [34] . Interestingly and in contrast to glycyrrhizin, NAC failed to inhibit H5N1 replication or H5N1-induced cytokine/chemokine expression in therapeutically relevant concentrations. Glycyrrhizin diminished H5N1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with H5N1 replication although redox-sensitive signalling pathways have been described to be involved in both processes.", "Glycyrrhizin diminished H5N1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with H5N1 replication although redox-sensitive signalling pathways have been described to be involved in both processes. Therefore, H5N1-induced proinflammatory gene expression appears to be more sensitive to inhibition of ROS formation than H5N1 replication. Indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] .", "Indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] . In a previous report, we could show that similar glycyrrhizin concentrations like those investigated here interfered with H5N1-induced pro-inflammatory gene expression but not with H5N1 replication in human monocyte-derived macrophages [57] . In addition, other immunomodulatory treatment regimens that did not influence H5N1 replication reduced mortality in H5N1-infected mice [31, 58] .", "In addition, other immunomodulatory treatment regimens that did not influence H5N1 replication reduced mortality in H5N1-infected mice [31, 58] . Therefore, glycyrrhizin represents a potential additional treatment option that interfers with both H5N1 replication and H5N1induced expression of pro-inflammatory cytokines in lung cells. Interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8 + T-lymphocytes.", "Interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8 + T-lymphocytes. Global immunosuppressants like corticosteroids failed to protect from lethal influenza virus infection [59] . Moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper NK cell cytolytic activity [60] .", "Moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper NK cell cytolytic activity [60] . In this context, glycyrrhizin had already been shown not to affect natural killer cell activity in the concentrations used here [57] . In conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation SNMC) interfere with highly pathogenic H5N1 influenza A virus replication and H5N1-induced proinflammatory gene expression at least in part through interference with H5N1-induced ROS formation and in turn reduced activation of p38, JNK, and NFkB in lung cells.", "In conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation SNMC) interfere with highly pathogenic H5N1 influenza A virus replication and H5N1-induced proinflammatory gene expression at least in part through interference with H5N1-induced ROS formation and in turn reduced activation of p38, JNK, and NFkB in lung cells. Since we used the clinical formulation SNMC effects of other ingredients like glycin or cystein cannot be excluded. Vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza A virus pandemic [61] .", "Vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza A virus pandemic [61] . Anti-inflammatory and immunomodulatory agents are considered to be important candidates as constituents of anti-influenza treatment strategies that may save lives in an influenza pandemic situation [61] . Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1-caused disease." ]

[ "Glycyrrhizin is known to exert antiviral and anti-inflammatory effects. Here, the effects of an approved parenteral glycyrrhizin preparation (Stronger Neo-Minophafen C) were investigated on highly pathogenic influenza A H5N1 virus replication, H5N1-induced apoptosis, and H5N1-induced pro-inflammatory responses in lung epithelial (A549) cells. Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher).", "Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher). Glycyrrhizin also diminished monocyte migration towards supernatants of H5N1-infected A549 cells. The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen species and (in turn) reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication.", "The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen species and (in turn) reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication. Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease. Text: Highly pathogenic H5N1 influenza A viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] .", "Text: Highly pathogenic H5N1 influenza A viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] . At least for the first wave of an H5N1 pandemic, no sufficient amounts of adequate vaccines will be available [1] [2] [3] [4] [6] [7] [8] . Therefore, antiviral therapy for influenza A viruses including highly pathogenic H5N1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] .", "Therefore, antiviral therapy for influenza A viruses including highly pathogenic H5N1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] . The neuraminidase inhibitors oseltamivir and zanamivir as well as the adamantanes amantadin and rimantadin that interfere with the influenza M2 protein are licensed for the treament of influenza [1] [2] [3] [4] 6] . However, the use of both drug classes is limited by the emergence of resistant virus strains.", "However, the use of both drug classes is limited by the emergence of resistant virus strains. In seasonal influenza strains, the majority of H3N2 viruses and a great proportion of H1N1 viruses in humans are now considered to be amantadine-and rimantadine-resistant [10] [11] [12] [13] . Moreover, a drastic increase in oseltamivir-resistant H1N1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] .", "Moreover, a drastic increase in oseltamivir-resistant H1N1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] . Preliminary data from the United States predict a further rise for the 2008/2009 season, possibly resulting in more than 90% of the circulating H1N1 strains to be oseltamivir resistant [14] . H5N1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza A virus strains and treatment-resistant H5N1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] .", "H5N1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza A virus strains and treatment-resistant H5N1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] . More-over, parenteral agents for the treatment of seriously ill patients are missing. Glycyrrhizin, a triterpene saponine, is a constituent of licorice root.", "Glycyrrhizin, a triterpene saponine, is a constituent of licorice root. It has been found to interfere with replication and/or cytopathogenic effect (CPE) induction of many viruses including respiratory viruses such as respiratory syncytial virus, SARS coronavirus, HIV, and influenza viruses [22] [23] [24] [25] [26] [27] [28] . Moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] .", "Moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] . The severity of human H5N1 disease has been associated with hypercytokinaemia (''cytokine storm'') [29, 30] . Delayed antiviral plus immunomodulator treatment reduced H5N1-induced mortality in mice [31] .", "Delayed antiviral plus immunomodulator treatment reduced H5N1-induced mortality in mice [31] . Therefore, antiinflammatory and immunomodulatory effects exerted by glycyrrhizin may be beneficial for treatment of H5N1. Also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza A virus replication and virus-induced pro-inflammatory responses [32] [33] [34] .", "Also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza A virus replication and virus-induced pro-inflammatory responses [32] [33] [34] . Stronger Neo-Minophagen C (SNMC) is a glycyrrhizin preparation (available as tablets or parenteral formulation) that is approved in Japan for the treatment of chronic hepatic diseases and is marketed in Japan, China, Korea, Taiwan, Indonesia, India, and Mongolia. Here, we investigated the influence of SNMC on H5N1 replication, on H5N1-induced cytokine expression, on H5N1-induced cellular oxidative stress, and on critical H5N1-induced cellular signalling events in human pneumocytes (A549 cell line).", "Here, we investigated the influence of SNMC on H5N1 replication, on H5N1-induced cytokine expression, on H5N1-induced cellular oxidative stress, and on critical H5N1-induced cellular signalling events in human pneumocytes (A549 cell line). Glycyrrhizin (Stronger Neo Minophagen C) was obtained from Minophagen Pharmaceuticals Co., Ltd. (Tokyo, Japan). The influenza strain A/Vietnam/1203/04 (H5N1) was received from the WHO Influenza Centre (National Institute for Medical Research, London, UK).", "The influenza strain A/Vietnam/1203/04 (H5N1) was received from the WHO Influenza Centre (National Institute for Medical Research, London, UK). The H5N1 influenza strain A/Thailand/ 1(Kan-1)/04 was obtained from Prof. Pilaipan Puthavathana (Mahidol University, Bangkok, Thailand). Virus stocks were prepared by infecting Vero cells (African green monkey kidney; ATCC, Manassas, VA) and aliquots were stored at 280uC.", "Virus stocks were prepared by infecting Vero cells (African green monkey kidney; ATCC, Manassas, VA) and aliquots were stored at 280uC. Virus titres were determined as 50% tissue culture infectious dose (TCID 50 /ml) in confluent Vero cells in 96-well microtiter plates. A549 cells (human lung carcinoma; ATCC: CCL-185, obtained from LGC Standards GmbH, Wesel, Germany) were grown at 37uC in minimal essential medium (MEM) supplemented with 10% FBS, 100 IU/ml of penicillin and 100 mg/ml streptomycin.", "A549 cells (human lung carcinoma; ATCC: CCL-185, obtained from LGC Standards GmbH, Wesel, Germany) were grown at 37uC in minimal essential medium (MEM) supplemented with 10% FBS, 100 IU/ml of penicillin and 100 mg/ml streptomycin. Human monocytes were isolated from buffy coats of healthy donors, obtained from Institute of Transfusion Medicine and Immune Haematology, German Red Cross Blood Donor Center, Johann Wolfgang Goethe-University, Frankfurt am Main. After centrifugation on Ficoll (Biocoll)-Hypaque density gradient (Biochrom AG, Berlin, Germany), mononuclear cells were collected from the interface and washed with PBS.", "After centrifugation on Ficoll (Biocoll)-Hypaque density gradient (Biochrom AG, Berlin, Germany), mononuclear cells were collected from the interface and washed with PBS. Then, monocytes were isolated using magnetically labeled CD14 MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) following the manufacturer's instructions. Monocytes were cultivated in IMDM supplemented with 10% pooled human serum, 100 IU/ml of penicillin, and 100 mg/ml streptomycin.", "Monocytes were cultivated in IMDM supplemented with 10% pooled human serum, 100 IU/ml of penicillin, and 100 mg/ml streptomycin. The cellular viability was assessed on confluent cell layers with CellTiter-GloH Luminescent Cell Viability Assay (Promega GmbH, Mannheim, Germany) according to the manufacturers' protocol. Cell viability was expressed as percentage of non-treated control.", "Cell viability was expressed as percentage of non-treated control. To determine intracellular NP localisation, H5N1-infected A549 were fixed 8 hours p.i. for 15 min with ice-cold acetone/ methanol (40:60, Mallinckrodt Baker B.V., Deventer, The Netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in PBS) directed against the influenza A virus nucleoprotein (NP) (Millipore, Molsheim, France).", "for 15 min with ice-cold acetone/ methanol (40:60, Mallinckrodt Baker B.V., Deventer, The Netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in PBS) directed against the influenza A virus nucleoprotein (NP) (Millipore, Molsheim, France). An Alexa Fluor 488 goat anti-mouse IgG (H&L) (Invitrogen, Eugene, Oregon, USA) was used (1 h incubation, 1:1000 in PBS) as secondary antibody. Nuclei were stained using 49,6-diamidino-2phenylindole (DAPI) (Sigma-Aldrich Chemie GmbH, Munich, Germany).", "Nuclei were stained using 49,6-diamidino-2phenylindole (DAPI) (Sigma-Aldrich Chemie GmbH, Munich, Germany). Fluorescence was visualised using Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany). For flow cytometric analysis, the same antibodies were used. The cytopathogenic effect (CPE) reduction assay was performed as described before [34] .", "The cytopathogenic effect (CPE) reduction assay was performed as described before [34] . Confluent A549 cell monolayers grown in 96-well microtitre plates were infected with influenza A strains at the indicated multiplicities of infection (MOIs). After a one hour adsorption period, cells were washed to remove non-detached virus.", "After a one hour adsorption period, cells were washed to remove non-detached virus. The virus-induced CPE was recorded at 24 h post infection (p.i.). Unless otherwise stated, A549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period.", "Unless otherwise stated, A549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period. For time-ofaddition experiments, glycyrrhizin was added exclusively during the 1 h pre-incubation period, exclusively during the 1 h adsorption period, or after exclusively after the wash-out of input virus. Total RNA was isolated from cell cultures using TRI reagent (Sigma-Aldrich, Munich, Germany).", "Total RNA was isolated from cell cultures using TRI reagent (Sigma-Aldrich, Munich, Germany). Real time PCR for H5 was performed using described methods [35] . The following primers were used: sense 59 acg tat gac tac ccg cag tat tca g 39; antisense 59 aga cca gcy acc atg att gc 39; probe 6-FAM-tca aca gtg gcg agt tcc cta gca-TAMRA.", "The following primers were used: sense 59 acg tat gac tac ccg cag tat tca g 39; antisense 59 aga cca gcy acc atg att gc 39; probe 6-FAM-tca aca gtg gcg agt tcc cta gca-TAMRA. The fraction of cells with fractional DNA content (''sub-G1'' cell subpopulation) indicates cytotoxicity. Sub-G1 cells are considered to be dead (usually apoptotic) cells.", "Sub-G1 cells are considered to be dead (usually apoptotic) cells. Cells were fixed with 70% ethanol for two hours at 220uC. The cellular DNA was stained using propidium iodide (20 mg/ml) and analysed by flow cytometry (FacsCalibur, BD Biosciences, Heidelberg, Germany).", "The cellular DNA was stained using propidium iodide (20 mg/ml) and analysed by flow cytometry (FacsCalibur, BD Biosciences, Heidelberg, Germany). Caspase activation was measured using the Caspase-Glo 8, 9, or 3/7 Assays (Promega, Mannheim, Germany) following the manufacturer's instructions. Cell culture supernatants were collected and frozen at 280uC.", "Cell culture supernatants were collected and frozen at 280uC. Cytokines/chemokines were quantified by specific ELISA Duo Sets (R&D Systems GmbH, Wiesbaden, Germany) following the manufacturer's instructions. NFkB activity was investigated in H5N1 (MOI 0.01)-infected cells by quantification of the NFkB subunits Rel A (p65) and NFkB1 (p50) from nuclear extracts using the TransAM TM transcription factor DNA-binding ELISAs (Active Motif, Rixensart, Belgium).", "NFkB activity was investigated in H5N1 (MOI 0.01)-infected cells by quantification of the NFkB subunits Rel A (p65) and NFkB1 (p50) from nuclear extracts using the TransAM TM transcription factor DNA-binding ELISAs (Active Motif, Rixensart, Belgium). Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction. Cell culture supernatants were investigated for chemotactic activity by measurement of the activity to induce monocyte migration through membrane inserts in 24-well plates (pore size 8 mm; BD Biosciences, Heidelberg, Germany).", "Cell culture supernatants were investigated for chemotactic activity by measurement of the activity to induce monocyte migration through membrane inserts in 24-well plates (pore size 8 mm; BD Biosciences, Heidelberg, Germany). Monocytes (1610 6 in 100 ml of IMDM with 10% pooled human serum) were added into the cell culture inserts (upper chamber) and cell culture supernatants (300 ml), were added to the lower chamber of the well. After a 48 h incubation period, cells were fixed with 4% paraformaldehyde and permeabilised with PBS containing 0.3% Tritron X-100.", "After a 48 h incubation period, cells were fixed with 4% paraformaldehyde and permeabilised with PBS containing 0.3% Tritron X-100. Then, nuclei were stained with 49,6-diamidino-2phenylindole (DAPI). The upper side of the membrane was wiped with a wet swab to remove the cells, while the lower side of the membrane was rinsed with PBS.", "The upper side of the membrane was wiped with a wet swab to remove the cells, while the lower side of the membrane was rinsed with PBS. The number of cells at the lower side of each membrane was quantified by counting of cells from three randomly chosen sections (3.7 mm 2 ) using an Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany). Cells were lysed in Triton X-sample buffer and separated by SDS-PAGE.", "Cells were lysed in Triton X-sample buffer and separated by SDS-PAGE. Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction. Proteins were detected using specific antibodies against bactin (Sigma-Aldrich Chemie GmbH, Munich, Germany), JNK, phosphorylated JNK, p38, or phosphorylated p38, (all purchased from New England Biolabs GmbH, Frankfurt am Main, Germany) and were visualised by enhanced chemiluminescence using a commercially available kit (Amersham, Freiburg, Germany).", "Proteins were detected using specific antibodies against bactin (Sigma-Aldrich Chemie GmbH, Munich, Germany), JNK, phosphorylated JNK, p38, or phosphorylated p38, (all purchased from New England Biolabs GmbH, Frankfurt am Main, Germany) and were visualised by enhanced chemiluminescence using a commercially available kit (Amersham, Freiburg, Germany). Reactive oxygen species (ROS) were detected using the Image-iT LIVE Green Reactive Oxygen Species Kit (Molecular Probes, distributed by Invitrogen, Karlsruhe, Germany). Two groups were compared by t-test.", "Two groups were compared by t-test. More groups were compared by ANOVA with subsequent Student-Newman-Keuls test. The A549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type II pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g.", "The A549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type II pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g. 6,37,38]. If not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period.", "If not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period. Glycyrrhizin 200 mg/ml (the maximum tested concentration) did not affect A549 cell viability (data not shown) but clearly decreased CPE formation in A549 cells infected with the H5N1 influenza strain A/Thailand/1(Kan-1)/04 at MOIs of 0.01, 0.1 or 1 ( Figure 1A ). Similar results were obtained in A549 cells infected with strain A/Vietnam/1203/04 (H5N1) (Suppl.", "Similar results were obtained in A549 cells infected with strain A/Vietnam/1203/04 (H5N1) (Suppl. Figure 1A) . Staining of A549 cells for influenza A nucleoprotein 24 h after infection with strain H5N1 A/Thailand/1(Kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza A nucleoprotein positive cells ( Figure 1B) .", "Staining of A549 cells for influenza A nucleoprotein 24 h after infection with strain H5N1 A/Thailand/1(Kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza A nucleoprotein positive cells ( Figure 1B) . To examine the influence of glycyrrhizin on virus progeny, A549 cells were infected with the H5N1 influenza strain A/ Thailand/1(Kan-1)/04 at MOI 0.01 or MOI 1 and infectious virus titres were determined 24 h post infection ( Figure 1C ). While glycyrrhizin in concentrations up to 50 mg/ml did not affect H5N1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (MOI 0.01: 13-fold reduction, MOI 1: 10-fold reduction).", "While glycyrrhizin in concentrations up to 50 mg/ml did not affect H5N1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (MOI 0.01: 13-fold reduction, MOI 1: 10-fold reduction). Next, influence of glycyrrhizin on H5N1 replication was confirmed by the detection of viral (H5) RNA using quantitative PCR. Only glycyrrhizin concentrations $100 mg/ml significantly reduced Figure 1B) or H5N1 A/Vietnam/1203/04-infected (Suppl.", "Only glycyrrhizin concentrations $100 mg/ml significantly reduced Figure 1B) or H5N1 A/Vietnam/1203/04-infected (Suppl. Figure 1C ) A549 cells (MOI 0.01) 24 h post infection. Time-of-addition experiments revealed that maximal effects were achieved when glycyrrhizin was continuously present starting with a 1 h pre-incubation period ( Figure 1D ).", "Time-of-addition experiments revealed that maximal effects were achieved when glycyrrhizin was continuously present starting with a 1 h pre-incubation period ( Figure 1D ). Addition of glycyrrhizin post infection showed reduced antiviral effects while pre-incubation alone or glycyrrhizin addition during the adsorption period did not significantly affect H5N1 replication. For investigation of H5N1-induced cytokine expression, five pro-inflammatory genes were chosen that had been correlated to severity of influenza disease: CXCL10 (also known as interferon-cinducible protein 10, IP-10), interleukin 6 (IL6), interleukin 8, (IL8; also known as CXCL8), CCL2 (also known as monocyte chemoattractant protein 1, MCP-1), and CCL5 (also known as RANTES).", "For investigation of H5N1-induced cytokine expression, five pro-inflammatory genes were chosen that had been correlated to severity of influenza disease: CXCL10 (also known as interferon-cinducible protein 10, IP-10), interleukin 6 (IL6), interleukin 8, (IL8; also known as CXCL8), CCL2 (also known as monocyte chemoattractant protein 1, MCP-1), and CCL5 (also known as RANTES). A549 cells were infected with H5N1 A/Thailand/ 1(Kan-1)/04 or H5N1 A/Vietnam/1203/04 at MOI 0.01, 0.1, or 1. Glycyrrhizin treatment was performed with 25, 50, 100, or 200 mg/ml.", "Glycyrrhizin treatment was performed with 25, 50, 100, or 200 mg/ml. Cytokine expression was detected 24 h post infection by ELISA. Glycyrrhizin did not affect cytokine expression of noninfected cells (data not shown) but inhibited expression of all cytokines investigated in H5N1-infected cells in a dose-dependent manner (Figure 2, Figure 3A ).", "Glycyrrhizin did not affect cytokine expression of noninfected cells (data not shown) but inhibited expression of all cytokines investigated in H5N1-infected cells in a dose-dependent manner (Figure 2, Figure 3A ). Effects were more pronounced at lower MOIs. Notably, expression of all cytokines except IL8 was significantly inhibited after treatment with glycyrrhizin 50 mg/ml Figure 3A ) although these glycyrrhizin concentrations had no effect on H5N1 replication in A549 cells (Figure 1, Figure S1 ).", "Notably, expression of all cytokines except IL8 was significantly inhibited after treatment with glycyrrhizin 50 mg/ml Figure 3A ) although these glycyrrhizin concentrations had no effect on H5N1 replication in A549 cells (Figure 1, Figure S1 ). Cytokine expression by influenza A virus-infected respiratory cells causes recruitment of peripheral blood monocytes into the lungs of patients where they differentiate to macrophages which are thought to contribute to influenza A virus pathogenicity [5, 39] . In a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.1)-infected A549 cells through 8 mm filters.", "In a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.1)-infected A549 cells through 8 mm filters. Monocyte migration towards supernatants of H5N1-infected cells was strongly increased relative to migration towards supernatants of non-infected cells. Treatment of H5N1- infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( Figure 3B ).", "Treatment of H5N1- infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( Figure 3B ). Influenza viruses including H5N1 have been shown to induce caspase-dependent apoptosis in airway cells and this apoptosis has been correlated to the virus pathogenicity [40, 41] . Glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( Figure 4A-C) .", "Glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( Figure 4A-C) . Glycyrrhizin concentrations $100 mg/ml inhibited H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.01)-induced activation of the initiator caspases 8 and 9 as well as of the effector caspases 3/7 in A549 cells as determined 24 h post infection ( Figure 4A-C) . Lower glycyrrhizin concentrations did not affect H5N1-induced apoptosis.", "Lower glycyrrhizin concentrations did not affect H5N1-induced apoptosis. The detection of cells in sub-G1 phase resulted in similar findings ( Figure 4D ). Substances that inhibit H5N1-induced caspase 3 activation including caspase 3 inhibitors cause nuclear retention of RNP complexes [34, 42] .", "Substances that inhibit H5N1-induced caspase 3 activation including caspase 3 inhibitors cause nuclear retention of RNP complexes [34, 42] . In accordance, glycyrrhizin also interfered with nuclear export RNP at MOI 1 ( Figure S2 ). Similar results were obtained in MOI 0.01 H5N1 A/Thailand/1(Kan-1)/04infected cells ( Figure S3 ).", "Similar results were obtained in MOI 0.01 H5N1 A/Thailand/1(Kan-1)/04infected cells ( Figure S3 ). Influence of glycyrrhizin on H5N1-induced activation of nuclear factor kB (NFkB), p38, and on H5N1-induced cellular reactive oxygen species (ROS) formation Activation of NFkB, p38, and JNK have been associated with influenza A virus replication and virus-induced pro-inflammatory gene expression [34, [43] [44] [45] [46] [47] . While glycyrrhizin did not influence NFkB activity in non-infected A549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited NFkB activation in H5N1-infected cells ( Figure 5A ).", "While glycyrrhizin did not influence NFkB activity in non-infected A549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited NFkB activation in H5N1-infected cells ( Figure 5A ). Moreover, glycyrrhizin inhibited H5N1-induced phosphorylation of the MAPKs p38 and JNK ( Figure 5B ). In addition to their roles during influenza A virus replication and virus-induced cytokine/chemokine expression, NFkB, p38, and JNK are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] .", "In addition to their roles during influenza A virus replication and virus-induced cytokine/chemokine expression, NFkB, p38, and JNK are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] . Antioxidants had been already found to interfere with influenza A virus-induced signalling through NFkB, p38, and JNK, with influenza A virus replication, and with influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] . Since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with H5N1-induced ROS formation.", "Since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with H5N1-induced ROS formation. Indeed glycyrrhizin exerted clear antioxidative effects in H5N1 (MOI 0.01)-infected cells ( Figure 5C ) causing significant reduction of ROS formation already at a concentration of 25 mg/ml ( Figure 5D ). Here, we show that glycyrrhizin inhibits the replication of highly pathogenic H5N1 influenza A virus, H5N1-induced apoptosis, and H5N1-induced expression of pro-inflammatory cytokines in lung-derived A549 cells.", "Here, we show that glycyrrhizin inhibits the replication of highly pathogenic H5N1 influenza A virus, H5N1-induced apoptosis, and H5N1-induced expression of pro-inflammatory cytokines in lung-derived A549 cells. After intravenous administration, achievable plasma concentrations of glycyrrhizin have been described to be about 100 mg/ml [52] . Therefore, the glycyrrhizin concentrations found to interfere with H5N1 replication and H5N1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels.", "Therefore, the glycyrrhizin concentrations found to interfere with H5N1 replication and H5N1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels. Notably, although higher glycyrrhizin concentrations were needed to interfere with SARS coronavirus replication [22] than with H5N1 replication, beneficial results were reported in glycyrrhizin (SNMC)-treated SARS patients in comparison to SARS patients who did not receive glycyrrhizin [23] . Notably, investigation of different glycyrrhizin derivatives against SARS coronavirus led to the identification of compounds with enhanced antiviral activity [53] .", "Notably, investigation of different glycyrrhizin derivatives against SARS coronavirus led to the identification of compounds with enhanced antiviral activity [53] . Therefore, glycyrrhizin might also serve as lead structure for the development of novel anti-influenza drugs. Experimental results suggested that glycyrrhizin might be able to affect seasonal influenza A virus disease by antiviral and immunomodulatory effects [26, 27] .", "Experimental results suggested that glycyrrhizin might be able to affect seasonal influenza A virus disease by antiviral and immunomodulatory effects [26, 27] . Mice were prevented from lethal H2N2 infection by glycyrrhizin although no influence on virus replication was detected. The mechanism was suggested to be induction of interferon-c in T-cells by glycyrrhizin [54] .", "The mechanism was suggested to be induction of interferon-c in T-cells by glycyrrhizin [54] . Moreover, glycyrrhizin was shown to influence seasonal influenza A virus replication through interaction with the cell membrane [25, 28] . However, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period.", "However, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period. Since glycyrrhizin addition during the adsorption period did not influence H5N1 replication in our experiments it appears not likely that membrane effects contribute to anti-H5N1 effects detected here in lower concentrations. Our results rather suggest that glycyrrhizin interferes with H5N1-induced oxidative stress.", "Our results rather suggest that glycyrrhizin interferes with H5N1-induced oxidative stress. Influenza A virus (including H5N1) infection induces ROS formation. Antioxidants were found to inhibit influenza A virus replication and influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] .", "Antioxidants were found to inhibit influenza A virus replication and influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] . Here, glycyrrhizin interfered with H5N1-induced activation of NFkB, p38, and JNK representing redox-sensitive signalling events [48] [49] [50] [51] involved in influenza A virus replication and influenza A virusinduced cellular cytokine/chemokine production [34, [43] [44] [45] [46] 55] . Glycyrrhizin 50 mg/ml significantly reduced H5N1-induced activation of NFkB.", "Glycyrrhizin 50 mg/ml significantly reduced H5N1-induced activation of NFkB. In addition, glycyrrhizin concentrations as low as 25 mg/ml effectively interfered with H5N1-induced ROS formation and with phosphorylation of the redox-sensitive MAPKs p38 and JNK. In our model, activation of p38 appears to be critical for H5N1-associated redox signalling since p38 inhibition had been shown before to mimick effects of the antioxidant N-acetyl-cysteine (NAC) [34] .", "In our model, activation of p38 appears to be critical for H5N1-associated redox signalling since p38 inhibition had been shown before to mimick effects of the antioxidant N-acetyl-cysteine (NAC) [34] . Interestingly and in contrast to glycyrrhizin, NAC failed to inhibit H5N1 replication or H5N1-induced cytokine/chemokine expression in therapeutically relevant concentrations. Glycyrrhizin diminished H5N1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with H5N1 replication although redox-sensitive signalling pathways have been described to be involved in both processes.", "Glycyrrhizin diminished H5N1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with H5N1 replication although redox-sensitive signalling pathways have been described to be involved in both processes. Therefore, H5N1-induced proinflammatory gene expression appears to be more sensitive to inhibition of ROS formation than H5N1 replication. Indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] .", "Indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] . In a previous report, we could show that similar glycyrrhizin concentrations like those investigated here interfered with H5N1-induced pro-inflammatory gene expression but not with H5N1 replication in human monocyte-derived macrophages [57] . In addition, other immunomodulatory treatment regimens that did not influence H5N1 replication reduced mortality in H5N1-infected mice [31, 58] .", "In addition, other immunomodulatory treatment regimens that did not influence H5N1 replication reduced mortality in H5N1-infected mice [31, 58] . Therefore, glycyrrhizin represents a potential additional treatment option that interfers with both H5N1 replication and H5N1induced expression of pro-inflammatory cytokines in lung cells. Interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8 + T-lymphocytes.", "Interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8 + T-lymphocytes. Global immunosuppressants like corticosteroids failed to protect from lethal influenza virus infection [59] . Moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper NK cell cytolytic activity [60] .", "Moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper NK cell cytolytic activity [60] . In this context, glycyrrhizin had already been shown not to affect natural killer cell activity in the concentrations used here [57] . In conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation SNMC) interfere with highly pathogenic H5N1 influenza A virus replication and H5N1-induced proinflammatory gene expression at least in part through interference with H5N1-induced ROS formation and in turn reduced activation of p38, JNK, and NFkB in lung cells.", "In conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation SNMC) interfere with highly pathogenic H5N1 influenza A virus replication and H5N1-induced proinflammatory gene expression at least in part through interference with H5N1-induced ROS formation and in turn reduced activation of p38, JNK, and NFkB in lung cells. Since we used the clinical formulation SNMC effects of other ingredients like glycin or cystein cannot be excluded. Vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza A virus pandemic [61] .", "Vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza A virus pandemic [61] . Anti-inflammatory and immunomodulatory agents are considered to be important candidates as constituents of anti-influenza treatment strategies that may save lives in an influenza pandemic situation [61] . Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1-caused disease." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU.", "METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia.", "Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived.", "It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support.", "Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS.", "Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children.", "Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] .", "Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] .", "In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs.", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series.", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled.", "From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis.", "Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] .", "Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies.", "Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.", "The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range). During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013.", "Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China.", "All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia.", "After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52).", "All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range.", "White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts (Table 2) . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ).", "CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ).", "Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days). All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples.", "In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay.", "Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ). Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients.", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip).", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) .", "Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived.", "Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B.", "(1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS.", "(2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients.", "(4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] .", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] .", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] .", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations.", "Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] .", "reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] .", "HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV.", "In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy.", "It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU.", "METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia.", "Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived.", "It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support.", "Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS.", "Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children.", "Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] .", "Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] .", "In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs.", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series.", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled.", "From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis.", "Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] .", "Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies.", "Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.", "The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range). During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013.", "Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China.", "All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia.", "After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52).", "All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range.", "White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts (Table 2) . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ).", "CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ).", "Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days). All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples.", "In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay.", "Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ). Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients.", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip).", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) .", "Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived.", "Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B.", "(1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS.", "(2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients.", "(4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] .", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] .", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] .", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations.", "Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] .", "reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] .", "HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV.", "In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy.", "It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU.", "METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia.", "Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived.", "It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support.", "Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS.", "Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children.", "Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] .", "Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] .", "In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs.", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series.", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled.", "From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis.", "Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] .", "Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies.", "Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.", "The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range). During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013.", "Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China.", "All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia.", "After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52).", "All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range.", "White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts (Table 2) . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ).", "CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ).", "Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days). All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples.", "In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay.", "Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ). Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients.", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip).", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) .", "Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived.", "Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B.", "(1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS.", "(2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients.", "(4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] .", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] .", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] .", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations.", "Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] .", "reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] .", "HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV.", "In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy.", "It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU.", "METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia.", "Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived.", "It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support.", "Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS.", "Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children.", "Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] .", "Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] .", "In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs.", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series.", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled.", "From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis.", "Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] .", "Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies.", "Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.", "The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range). During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013.", "Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China.", "All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia.", "After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52).", "All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range.", "White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts (Table 2) . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ).", "CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ).", "Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days). All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples.", "In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay.", "Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ). Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients.", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip).", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) .", "Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived.", "Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B.", "(1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS.", "(2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients.", "(4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] .", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] .", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] .", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations.", "Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] .", "reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] .", "HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV.", "In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy.", "It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU.", "METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia.", "Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived.", "It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support.", "Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS.", "Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children.", "Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] .", "Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] .", "In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs.", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series.", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled.", "From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis.", "Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] .", "Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies.", "Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.", "The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range). During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013.", "Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China.", "All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia.", "After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52).", "All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range.", "White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts (Table 2) . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ).", "CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ).", "Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days). All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples.", "In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay.", "Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ). Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients.", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip).", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) .", "Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived.", "Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B.", "(1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS.", "(2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients.", "(4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] .", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] .", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] .", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations.", "Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] .", "reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] .", "HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV.", "In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy.", "It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU.", "METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia.", "Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived.", "It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support.", "Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS.", "Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children.", "Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] .", "Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] .", "In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs.", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series.", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled.", "From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis.", "Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] .", "Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies.", "Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.", "The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range). During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013.", "Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China.", "All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia.", "After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52).", "All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range.", "White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts (Table 2) . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ).", "CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ).", "Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days). All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples.", "In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay.", "Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ). Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients.", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip).", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) .", "Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived.", "Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B.", "(1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS.", "(2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients.", "(4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] .", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] .", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] .", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations.", "Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] .", "reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] .", "HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV.", "In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy.", "It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU.", "METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia.", "Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived.", "It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support.", "Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS.", "Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children.", "Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] .", "Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] .", "In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs.", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series.", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled.", "From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis.", "Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] .", "Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies.", "Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.", "The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range). During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013.", "Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China.", "All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia.", "After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52).", "All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range.", "White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts (Table 2) . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ).", "CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ).", "Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days). All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples.", "In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay.", "Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ). Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients.", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip).", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) .", "Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived.", "Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B.", "(1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS.", "(2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients.", "(4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] .", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] .", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] .", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations.", "Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] .", "reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] .", "HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV.", "In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy.", "It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU.", "METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia.", "Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived.", "It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support.", "Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS.", "Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children.", "Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] .", "Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] .", "In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs.", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series.", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled.", "From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis.", "Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] .", "Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies.", "Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.", "The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range). During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013.", "Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China.", "All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia.", "After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52).", "All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range.", "White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts (Table 2) . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ).", "CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ).", "Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days). All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples.", "In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay.", "Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ). Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients.", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip).", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) .", "Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived.", "Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B.", "(1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS.", "(2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients.", "(4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] .", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] .", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] .", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations.", "Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] .", "reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] .", "HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV.", "In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy.", "It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU.", "METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia.", "Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived.", "It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support.", "Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS.", "Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children.", "Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] .", "Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] .", "In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs.", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series.", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled.", "From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis.", "Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] .", "Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies.", "Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.", "The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range). During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013.", "Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China.", "All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia.", "After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52).", "All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range.", "White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts (Table 2) . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ).", "CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ).", "Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days). All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples.", "In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay.", "Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ). Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients.", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip).", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) .", "Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived.", "Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B.", "(1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS.", "(2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients.", "(4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] .", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] .", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] .", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations.", "Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] .", "reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] .", "HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV.", "In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy.", "It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU.", "METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia.", "Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived.", "It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support.", "Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS.", "Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children.", "Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] .", "Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] .", "In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs.", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series.", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled.", "From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis.", "Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] .", "Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies.", "Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.", "The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range). During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013.", "Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China.", "All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia.", "After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52).", "All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range.", "White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts (Table 2) . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ).", "CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ).", "Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days). All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples.", "In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay.", "Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ). Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients.", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip).", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) .", "Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived.", "Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B.", "(1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS.", "(2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients.", "(4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] .", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] .", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] .", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations.", "Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] .", "reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] .", "HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV.", "In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy.", "It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU.", "METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia.", "Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived.", "It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support.", "Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS.", "Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children.", "Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] .", "Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] .", "In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs.", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series.", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled.", "From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis.", "Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] .", "Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies.", "Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.", "The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range). During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013.", "Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China.", "All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia.", "After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52).", "All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range.", "White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts (Table 2) . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ).", "CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ).", "Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days). All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples.", "In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay.", "Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ). Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients.", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip).", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) .", "Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived.", "Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B.", "(1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS.", "(2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients.", "(4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] .", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] .", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] .", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations.", "Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] .", "reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] .", "HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV.", "In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy.", "It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU.", "METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia.", "Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived.", "It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support.", "Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS.", "Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children.", "Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] .", "Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] .", "In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs.", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series.", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled.", "From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis.", "Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] .", "Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies.", "Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.", "The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range). During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013.", "Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China.", "All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia.", "After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52).", "All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range.", "White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts (Table 2) . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ).", "CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ).", "Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days). All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples.", "In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay.", "Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ). Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients.", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip).", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) .", "Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived.", "Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B.", "(1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS.", "(2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients.", "(4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] .", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] .", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] .", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations.", "Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] .", "reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] .", "HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV.", "In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy.", "It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU.", "METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia.", "Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived.", "It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support.", "Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS.", "Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children.", "Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] .", "Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] .", "In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs.", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series.", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled.", "From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis.", "Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] .", "Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies.", "Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.", "The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range). During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013.", "Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China.", "All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia.", "After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52).", "All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range.", "White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts (Table 2) . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ).", "CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ).", "Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days). All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples.", "In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay.", "Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ). Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients.", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip).", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) .", "Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived.", "Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B.", "(1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS.", "(2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients.", "(4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] .", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] .", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] .", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations.", "Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] .", "reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] .", "HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV.", "In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy.", "It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU.", "METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia.", "Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived.", "It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support.", "Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS.", "Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children.", "Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] .", "Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] .", "In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs.", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series.", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled.", "From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis.", "Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] .", "Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies.", "Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.", "The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range). During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013.", "Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China.", "All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia.", "After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52).", "All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range.", "White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts (Table 2) . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ).", "CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ).", "Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days). All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples.", "In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay.", "Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ). Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients.", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip).", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) .", "Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived.", "Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B.", "(1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS.", "(2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients.", "(4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] .", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] .", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] .", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations.", "Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] .", "reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] .", "HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV.", "In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy.", "It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU.", "METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia.", "Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived.", "It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support.", "Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS.", "Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children.", "Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] .", "Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] .", "In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs.", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series.", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled.", "From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis.", "Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] .", "Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies.", "Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.", "The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range). During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013.", "Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China.", "All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia.", "After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52).", "All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range.", "White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts (Table 2) . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ).", "CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ).", "Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days). All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples.", "In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay.", "Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ). Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients.", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip).", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) .", "Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived.", "Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B.", "(1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS.", "(2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients.", "(4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] .", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] .", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] .", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations.", "Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] .", "reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] .", "HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV.", "In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy.", "It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU.", "METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia.", "Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived.", "It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support.", "Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS.", "Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children.", "Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] .", "Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] .", "In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs.", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series.", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled.", "From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis.", "Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] .", "Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies.", "Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.", "The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range). During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013.", "Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China.", "All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia.", "After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52).", "All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range.", "White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts (Table 2) . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ).", "CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ).", "Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days). All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples.", "In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay.", "Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ). Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients.", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip).", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) .", "Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived.", "Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B.", "(1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS.", "(2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients.", "(4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] .", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] .", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] .", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations.", "Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] .", "reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] .", "HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV.", "In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy.", "It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU.", "METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia.", "Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived.", "It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support.", "Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS.", "Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children.", "Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] .", "Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] .", "In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs.", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series.", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled.", "From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis.", "Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] .", "Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies.", "Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.", "The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range). During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013.", "Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China.", "All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia.", "After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52).", "All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range.", "White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts (Table 2) . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ).", "CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ).", "Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days). All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples.", "In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay.", "Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ). Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients.", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip).", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) .", "Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived.", "Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B.", "(1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS.", "(2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients.", "(4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] .", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] .", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] .", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations.", "Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] .", "reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] .", "HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV.", "In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy.", "It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU.", "METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included.", "RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia.", "Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively.", "Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived.", "It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support.", "Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS.", "Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice.", "The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children.", "Registered 20 April 2012\n\nText: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] .", "Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] .", "In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs.", "Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] . Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series.", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU. Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled.", "From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis.", "Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Each patient gave written informed consent for their data to be used for research and publication. Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Concomitant medications, respiratory support, complications and outcomes were also recorded. Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] .", "Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies.", "Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.", "The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay. Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods. Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range). During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013.", "Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "The other two patients had mild ARDS and were infected with other types of HAdVs. All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China.", "All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission.", "Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative. Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients.", "Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia.", "After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52).", "All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range.", "White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range. Four patients had lower than normal T-cell subset counts (Table 2) . CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ).", "CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients.", "Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ).", "Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days). All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples.", "In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay.", "Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived.", "The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ). Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients.", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead.", "Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support. All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip).", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) .", "Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived.", "Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively. To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B.", "(1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS.", "(2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients.", "(4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support. Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] .", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] .", "It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS. The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] .", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic.", "They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region. Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations.", "Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] . Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] .", "reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted.", "Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] .", "HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.", "The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome. The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV.", "In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.", "For patients with severe ARDS, ECMO should be considered a better choice for oxygenation. Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy.", "It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ "INTRODUCTION: Community-acquired pneumonia (CAP) requires prompt treatment, but its diagnosis is complex. Improvement of bacterial CAP diagnosis by biomarkers has been evaluated using chest X-ray infiltrate as the CAP gold standard, producing conflicting results. We analyzed the diagnostic accuracy of biomarkers in suspected CAP adults visiting emergency departments for whom CAP diagnosis was established by an adjudication committee which founded its judgment on a systematic multidetector thoracic CT scan.", "We analyzed the diagnostic accuracy of biomarkers in suspected CAP adults visiting emergency departments for whom CAP diagnosis was established by an adjudication committee which founded its judgment on a systematic multidetector thoracic CT scan. METHODS: In an ancillary study of a multi-center prospective study evaluating the impact of systematic thoracic CT scan on CAP diagnosis, sensitivity and specificity of C-reactive protein (CRP) and procalcitonin (PCT) were evaluated. Systematic nasopharyngeal multiplex respiratory virus PCR was performed at inclusion.", "Systematic nasopharyngeal multiplex respiratory virus PCR was performed at inclusion. An adjudication committee classified CAP diagnostic probability on a 4-level Likert scale, based on all available data. RESULTS: Two hundred patients with suspected CAP were analyzed.", "RESULTS: Two hundred patients with suspected CAP were analyzed. The adjudication committee classified 98 patients (49.0 %) as definite CAP, 8 (4.0 %) as probable, 23 (11.5 %) as possible and excluded in 71 (35.5 %, including 29 patients with pulmonary infiltrates on chest X-ray). Among patients with radiological pulmonary infiltrate, 23 % were finally classified as excluded.", "Among patients with radiological pulmonary infiltrate, 23 % were finally classified as excluded. Viruses were identified by PCR in 29 % of patients classified as definite. Area under the curve was 0.787 [95 % confidence interval (95 % CI), 0.717 to 0.857] for CRP and 0.655 (95 % CI, 0.570 to 0.739) for PCT to detect definite CAP.", "Area under the curve was 0.787 [95 % confidence interval (95 % CI), 0.717 to 0.857] for CRP and 0.655 (95 % CI, 0.570 to 0.739) for PCT to detect definite CAP. CRP threshold at 50 mg/L resulted in a positive predictive value of 0.76 and a negative predictive value of 0.75. No PCT cut-off resulted in satisfactory positive or negative predictive values.", "No PCT cut-off resulted in satisfactory positive or negative predictive values. CRP and PCT accuracy was not improved by exclusion of the 25 (25.5 %) definite viral CAP cases. CONCLUSIONS: For patients with suspected CAP visiting emergency departments, diagnostic accuracy of CRP and PCT are insufficient to confirm the CAP diagnosis established using a gold standard that includes thoracic CT scan.", "CONCLUSIONS: For patients with suspected CAP visiting emergency departments, diagnostic accuracy of CRP and PCT are insufficient to confirm the CAP diagnosis established using a gold standard that includes thoracic CT scan. Diagnostic accuracy of these biomarkers is also insufficient to distinguish bacterial CAP from viral CAP. TRIAL REGISTRATION: ClinicalTrials.gov registry NCT01574066 (February 7, 2012) ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (.1186/s13054-015-1083-6) contains supplementary material, which is available to authorized users.", "TRIAL REGISTRATION: ClinicalTrials.gov registry NCT01574066 (February 7, 2012) ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (.1186/s13054-015-1083-6) contains supplementary material, which is available to authorized users. Text: Community-acquired pneumonia (CAP) is a frequently seen disease, with high morbidity and mortality, accounting for 600,000 hospitalizations each year. It represents the seventh leading cause of death in the USA [1] .", "It represents the seventh leading cause of death in the USA [1] . CAP prognosis depends on the rapidity of specific treatment, which should ideally be initiated within four hours and no later than eight hours after diagnosis [2, 3] . CAP diagnosis is based on the clustering of non-specific pulmonary and general symptoms [4, 5] , an increase in biomarkers reflecting systemic inflammatory response syndrome (SIRS), and the presence of new parenchymal infiltrates on chest X-ray.", "CAP diagnosis is based on the clustering of non-specific pulmonary and general symptoms [4, 5] , an increase in biomarkers reflecting systemic inflammatory response syndrome (SIRS), and the presence of new parenchymal infiltrates on chest X-ray. However, CAP diagnosis remains uncertain in many cases with alternative diagnoses, such as cardiac failure, acute bronchitis, chronic obstructive pulmonary disease (COPD) exacerbations, pulmonary embolism, neoplasia, and sepsis [6, 7] . Part of the uncertainty of CAP diagnosis may be due to the high rate of chest X-ray misdiagnosis [8, 9] ; over diagnosis of CAP is frequent when infiltrates of noninfectious origin coexist with pulmonary or general symptoms, and the diagnosis of CAP is often ignored when the lung infiltrates are at the limit of visibility or are hidden due to superposition [10] .", "Part of the uncertainty of CAP diagnosis may be due to the high rate of chest X-ray misdiagnosis [8, 9] ; over diagnosis of CAP is frequent when infiltrates of noninfectious origin coexist with pulmonary or general symptoms, and the diagnosis of CAP is often ignored when the lung infiltrates are at the limit of visibility or are hidden due to superposition [10] . We recently published a study in which thoracic CT scan was systematically performed in a population of clinically suspected CAP patients visiting the emergency department for CAP (the ESCAPED study) [11] . We showed that CAP diagnosis based on chest X-ray led to a false CAP diagnosis in many patients: among CAP suspected patients with radiological pulmonary infiltrate, CAP diagnosis was excluded in around 30 % of patients based on CT scan results; on the contrary, among patients without radiological pulmonary infiltrate, one-third had a pulmonary infiltrate on thoracic CT-scan.", "We showed that CAP diagnosis based on chest X-ray led to a false CAP diagnosis in many patients: among CAP suspected patients with radiological pulmonary infiltrate, CAP diagnosis was excluded in around 30 % of patients based on CT scan results; on the contrary, among patients without radiological pulmonary infiltrate, one-third had a pulmonary infiltrate on thoracic CT-scan. We also reported the isolation of viruses in one-third of patients [11, 12] . Several attempts have been made to improve CAP diagnosis based on biomarkers, such as C-reactive protein (CRP) and procalcitonin (PCT); however, there are conflicting data on their reliability [13] [14] [15] [16] [17] .", "Several attempts have been made to improve CAP diagnosis based on biomarkers, such as C-reactive protein (CRP) and procalcitonin (PCT); however, there are conflicting data on their reliability [13] [14] [15] [16] [17] . This could be due to the consideration of CAP diagnosis based on chest X-ray as establishing pulmonary infection. In the present study, we aimed to analyze CRP and PCT values in the population of the ESCAPED study reported above for whom CAP diagnosis was established by an adjudication committee which founded its judgment on all usual available data, systematic multidetector thoracic CT scan performed at inclusion, and results from a day-28 follow-up.", "In the present study, we aimed to analyze CRP and PCT values in the population of the ESCAPED study reported above for whom CAP diagnosis was established by an adjudication committee which founded its judgment on all usual available data, systematic multidetector thoracic CT scan performed at inclusion, and results from a day-28 follow-up. We also analyzed whether the viral etiology of definite CAP based on polymerase chain reaction (PCR) multiplex naso-pharyngeal swab interfered with the accuracy of the biomarkers. Setting ESCAPED was a multicenter, prospective, interventional study, entitled \"Early Thoracic CT-Scan for Community-Acquired Pneumonia at the Emergency Department (ESCAPED)\" [11] , conducted from November 2011 to January 2013, in four emergency departments (EDs) of four tertiary teaching hospitals in Paris, France, designed to measure the impact of thoracic CT scan on clinical decision.", "Setting ESCAPED was a multicenter, prospective, interventional study, entitled \"Early Thoracic CT-Scan for Community-Acquired Pneumonia at the Emergency Department (ESCAPED)\" [11] , conducted from November 2011 to January 2013, in four emergency departments (EDs) of four tertiary teaching hospitals in Paris, France, designed to measure the impact of thoracic CT scan on clinical decision. The study was sponsored and monitored by the Paris public health hospitals, and funded by the French Ministry of Health. The French health authorities (Agence nationale de sécurité des medicaments et produits de santé, ANSM) and the institutional review board for the protection of human subjects approved the study protocol and patient informed consent procedures.", "The French health authorities (Agence nationale de sécurité des medicaments et produits de santé, ANSM) and the institutional review board for the protection of human subjects approved the study protocol and patient informed consent procedures. All enrolled patients provided written informed consent for inclusion. The protocol was registered in the clinicaltrial.gov website under the PACSCAN acronym, the French translation of the English ESCAPED acronym (NCT01574066).", "The protocol was registered in the clinicaltrial.gov website under the PACSCAN acronym, the French translation of the English ESCAPED acronym (NCT01574066). The Ethics Committee of Ile de France (Comité de Protection des Personnes. Paris N°2 011-oct-12749) approved the study protocol.", "Paris N°2 011-oct-12749) approved the study protocol. The primary objective was to compare CRP and PCT values in the four different categories of CAP level of certainty using the day-28 adjudication committee classification. The four categories were: 1) absence of CAP hereafter referred to as excluded CAP diagnosis; 2) possible CAP; 3) probable CAP; and 4) definite CAP.", "The four categories were: 1) absence of CAP hereafter referred to as excluded CAP diagnosis; 2) possible CAP; 3) probable CAP; and 4) definite CAP. The secondary objectives were to assess whether CRP and PCT were associated with CAP diagnosis using sensitivity analyses in three successive subgroups chosen a priori; 1) when specifically considering patients classified as having excluded CAP diagnosis and definite CAP (i.e., the patients for whom the level of certainty was the highest); 2) when patients with excluded CAP diagnosis and diagnosed extra-pulmonary infectious disease (which may increase biomarker values) were not taken into account, in the excluded CAP group; and 3) when patients classified as viral CAP were not taken into account in the definite CAP group, as PCT has been reported to be lower in viral infections as compared to bacterial infections [18] . Consecutive adults ( [19] .", "Consecutive adults ( [19] . Multidetector thoracic CT-scan was performed after chest X-ray, ideally within the four hours following inclusion. Chest X-ray and thoracic CT-scan were performed using a standardized protocol.", "Chest X-ray and thoracic CT-scan were performed using a standardized protocol. The four levels of CAP probability according to CT scan were defined as definite (systematic alveolar condensation, alveolar condensation with peripheral and localized ground glass opacities, bronchiolar focal or multifocal micronodules), probable (peripheral alveolar condensation, retractile systematic alveolar condensation, or diffuse ground glass opacities), possible (pulmonary infarct), or excluded (pulmonary mass, other abnormalities, or normal images). Scan views were recorded on a DVD.", "Scan views were recorded on a DVD. Based on data collected from baseline standardized case report forms, DVD recorded pictures of X-ray and CTscan, and blinded to local interpretations, an adjudication committee consisting of three independent senior experts in infectious diseases, pneumology and radiology retrospectively assigned the probability of CAP diagnosis using the same 4-level Likert scale, with all available data including patients' discharge summary, and follow-up data obtained by assistant investigators who contacted by phone either the patient, relatives or general practitioners at day 28. For this study, the gold standard of CAP was the diagnosis assessed by this adjudication committee.", "For this study, the gold standard of CAP was the diagnosis assessed by this adjudication committee. Alternative diagnoses were established for excluded CAP and classified as non-CAP pulmonary diseases and extra-pulmonary infectious diseases and others. Blood samples were collected at inclusion in sodium heparin-treated tubes, centrifuged, and stored at −40°C until completion of the study.", "Blood samples were collected at inclusion in sodium heparin-treated tubes, centrifuged, and stored at −40°C until completion of the study. CRP and PCT concentrations were measured a posteriori on plasma collection (see Additional file 1 for methodology), except for patients in whom marker dosage was performed by the emergency practitioner on his own initiative. Naso-pharyngeal swabs were collected at enrollment and placed in a Middle Virocult MWE (Sigma®) transport medium.", "Naso-pharyngeal swabs were collected at enrollment and placed in a Middle Virocult MWE (Sigma®) transport medium. Samples were kept at room temperature and sent to the virology laboratory of Bichat -Claude Bernard Hospital (Paris) as soon as possible after collection. The samples were not frozen and thawed.", "The samples were not frozen and thawed. Multiplex PCR (RespiFinder-19 assay (Pathofinder®, Maastricht, Netherlands)) was performed on naso-pharyngeal swabs to detect 15 respiratory viruses -coronavirus 229E, NL63, OC43, human metapneumovirus (hMPV), influenza A, A (H1N1) pdm2009 and B viruses, parainfluenza viruses 1, 2, 3, and 4, respiratory syncytial virus (RSV) A and B, rhinovirus, adenovirus, and 4 intracellular bacteria -Bordetella pertussis, Chlamydophila pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, in one reaction. The multiplex PCR results were not available to the adjudication committee.", "The multiplex PCR results were not available to the adjudication committee. Routine microbiological examinations were also performed at the discretion of the emergency physicians and included blood culture, sputum culture, and antigenuria (see Additional file 1 for methodology). CAP, classified as definite, was considered as being of viral origin when multiplex PCR was positive for at least one of the 15 respiratory viruses and no bacteria were found using PCR and routine bacterial microbiological samples (sputum, blood culture, antigenuria) when performed.", "CAP, classified as definite, was considered as being of viral origin when multiplex PCR was positive for at least one of the 15 respiratory viruses and no bacteria were found using PCR and routine bacterial microbiological samples (sputum, blood culture, antigenuria) when performed. Baseline and follow-up characteristics were described by means and standard deviations (SD) or by median and interquartile range (IQR) for continuous variables normally distributed or with skewed distribution, respectively, and by percentages for categorical variables, for the total study population and for the study groups. We performed chi-square or Fisher exact tests when appropriate for qualitative variables, and the Student or Mann-Whitney tests for continuous variables with skewed distributions to compare baseline patient characteristics and study outcomes between study groups.", "We performed chi-square or Fisher exact tests when appropriate for qualitative variables, and the Student or Mann-Whitney tests for continuous variables with skewed distributions to compare baseline patient characteristics and study outcomes between study groups. The distribution values of the biomarkers were determined in the different populations of patients using boxplots. The performances of CRP and PCT in predicting definite CAP were evaluated by sensitivity analysis (definite CAP vs excluded CAP).", "The performances of CRP and PCT in predicting definite CAP were evaluated by sensitivity analysis (definite CAP vs excluded CAP). CRP was evaluated at several cut-off points of 20 mg/L, 30 mg/L, 50 mg/L, 70 mg/L, and 100 mg/L, values used in previous studies [15, 20, 21] . Several cut-off points for PCT were chosen at the level of 0.10 μg/L [18] , and at the two levels for suspected bacterial infection as stated by the manufacturer, i.e., 0.25 μg/L and 0.50 μg/L.", "Several cut-off points for PCT were chosen at the level of 0.10 μg/L [18] , and at the two levels for suspected bacterial infection as stated by the manufacturer, i.e., 0.25 μg/L and 0.50 μg/L. Sensitivities, specificities, positive predictive values (PPVs), negative predictive values (NPVs), and likelihood ratio were calculated. Receiver operating characteristic (ROC) curves were drawn, area under the curve AUC was computed and optimal cut-off was identified by the maximization of the Youden's index, comparing biomarker values in patients with excluded CAP and definite CAP.", "Receiver operating characteristic (ROC) curves were drawn, area under the curve AUC was computed and optimal cut-off was identified by the maximization of the Youden's index, comparing biomarker values in patients with excluded CAP and definite CAP. From these optimal cut-offs for CRP and PCT, sensitivity analyses were performed combining the CRP and PCT cut-offs. A multivariate logistic regression model was built to identify factors associated with having definite CAP as compared to having an excluded CAP diagnosis.", "A multivariate logistic regression model was built to identify factors associated with having definite CAP as compared to having an excluded CAP diagnosis. We excluded from the excluded CAP diagnosis group, patients with an extra-pulmonary infectious disease. All variables with a p value of < 0.25 in the bivariate analysis were entered into a multivariate logistic regression with a backward stepwise approach; the discrimination was evaluated by the C-index and its 95 % confidence interval (95 % CI) and the calibration was evaluated by the Hosmer Lemeshow goodness-of-fit test.", "All variables with a p value of < 0.25 in the bivariate analysis were entered into a multivariate logistic regression with a backward stepwise approach; the discrimination was evaluated by the C-index and its 95 % confidence interval (95 % CI) and the calibration was evaluated by the Hosmer Lemeshow goodness-of-fit test. All tests were two-sided, and p-values below 0.05 were considered to denote statistical significance. All statistical analyses were performed using SPSS statistical software version 21.0 (SPSS Inc., Chicago, IL, USA).", "All statistical analyses were performed using SPSS statistical software version 21.0 (SPSS Inc., Chicago, IL, USA). Two hundred patients with suspected CAP out of the 319 in the ESCAPED study were included in the present study, for which CRP and PCT assays and nasopharyngeal swab for multiplex PCR were available (Fig. 1) .", "1) . Characteristics of the 200 patients (age, age more than 65, gender, probability of CAP diagnosis by adjudication committee) were not significantly different from those of the 119 other patients of the ESCAPED study and are summarized in Table 1 . CRP and PCT assays were performed based on the emergency practitioner's own initiative in 70 patients for CRP and 131 for PCT, or performed a posteriori on plasma samples of the remaining patients.", "CRP and PCT assays were performed based on the emergency practitioner's own initiative in 70 patients for CRP and 131 for PCT, or performed a posteriori on plasma samples of the remaining patients. Sex ratio was approximately 1. More than half of the patients (54 %) were 65 years of age or older.", "More than half of the patients (54 %) were 65 years of age or older. The \n\nPulmonary infiltrates were seen on chest X-ray in 127 (63.5 %) patients. Thoracic CT-scan excluded a CAP diagnosis in 16.5 % of these 127 patients; on the contrary, thoracic CT-scan revealed a parenchymal infiltrate in 27 % of the 73 patients without infiltrate on chest X-ray.", "Thoracic CT-scan excluded a CAP diagnosis in 16.5 % of these 127 patients; on the contrary, thoracic CT-scan revealed a parenchymal infiltrate in 27 % of the 73 patients without infiltrate on chest X-ray. Based on all available data including multidetector CT scan results (but excluding PCR results), the adjudication \n\nThe CRP and PCT distributions in the 200 patients are presented in Fig. 2 A statistically significant difference between the two groups (excluded CAP vs definite CAP) was demonstrated for several cut-off points for CRP and PCT ( Table 2 ).", "2 A statistically significant difference between the two groups (excluded CAP vs definite CAP) was demonstrated for several cut-off points for CRP and PCT ( Table 2 ). For CRP, the value of 50 mg/L resulted in a PPV of 0.76 and a NPV of 0.75. For PCT, no value resulted in a satisfactory PPV or NPV.", "For PCT, no value resulted in a satisfactory PPV or NPV. For these two biochemical markers, the ability to predict CAP was evaluated by a ROC curve. The AUC was 0.787 (95 % CI 0.717-0.857), optimal cut-off = 45.9 mg/L for CRP (Fig.", "The AUC was 0.787 (95 % CI 0.717-0.857), optimal cut-off = 45.9 mg/L for CRP (Fig. 3 ) and 0.655 (95 % CI 0.570-0.739), optimal cut-off = 0.13 μg/ L for PCT (Fig. 4) .", "4) . Sensitivity analyses for the combination of CRP and PCT, using these optimal cut-offs, resulted in a PPV of 0.74 and a NPV of 0.58. Use of the other PCT cut-offs did not result in better PPV or NPV ( Table 2) .", "Use of the other PCT cut-offs did not result in better PPV or NPV ( Table 2) . The present study is novel as patients prospectively benefited from extensive investigation to determine the diagnosis of CAP in the ED, including both early multidetector thoracic CT-scan and day-28 adjudication committee. This led to the correction of CAP diagnosis previously based on chest X-ray in a high number of patients.", "This led to the correction of CAP diagnosis previously based on chest X-ray in a high number of patients. In these extensively characterized patients, both CRP and PCT lacked operational precision to allow the decisionmaking process to rule out or confirm diagnosis of CAP even in selected subgroups. The clinical characteristics of the patients included in this sub-study are consistent with those in the current literature.", "The clinical characteristics of the patients included in this sub-study are consistent with those in the current literature. As previously reported, patients frequently had a history of respiratory disorders, cancer and congestive heart failure [21, 22] . The design of the ESCAPED study required exclusion of patients within the highest CRB 65 categories, which limited the inclusion of patients older than 65.", "The design of the ESCAPED study required exclusion of patients within the highest CRB 65 categories, which limited the inclusion of patients older than 65. This may explain why the mean age of our patients (64 years) falls within the lower values of those reported elsewhere [19] . Data to identify the microbial agent responsible for the disease were collected by the usual techniques and multiplex PCR.", "Data to identify the microbial agent responsible for the disease were collected by the usual techniques and multiplex PCR. Viral identification using naso-pharyngeal PCR that revealed viral respiratory infection in approximately one-third of cases was concordant with values reported in the literature [23] . Therefore, we believe that our results can be extrapolated to most emergency patients suffering from CAP.", "Therefore, we believe that our results can be extrapolated to most emergency patients suffering from CAP. In the present study, patients were recruited on the basis of initial clinical assessment for the diagnosis of CAP. Therefore, we believe that the characteristics of the patients closely correspond to those that lead practitioners to consider a possible diagnosis of CAP.", "Therefore, we believe that the characteristics of the patients closely correspond to those that lead practitioners to consider a possible diagnosis of CAP. In these patients, the design of our study allowed us to confirm or refute CAP diagnosis with a high level of certainty. Results confirmed the poor predictive value of clinical symptoms (new onset of systemic features and symptoms of an acute lower respiratory tract illness) in identifying CAP patients [21] .", "Results confirmed the poor predictive value of clinical symptoms (new onset of systemic features and symptoms of an acute lower respiratory tract illness) in identifying CAP patients [21] . Indeed, clinical presentation of excluded CAP patients was similar to that of definite CAP patients except for fever and cough that were more frequent in definite CAP patients. Furthermore, the design also revealed that the combination of clinical symptoms and chest X-ray results led to CAP misdiagnosis in a high number of patients, including the 98 whose CAP diagnosis was excluded by the adjudication committee and who would have been considered as possible, probable or definite CAP without the use of the CT scan.", "Furthermore, the design also revealed that the combination of clinical symptoms and chest X-ray results led to CAP misdiagnosis in a high number of patients, including the 98 whose CAP diagnosis was excluded by the adjudication committee and who would have been considered as possible, probable or definite CAP without the use of the CT scan. This low specificity of clinical-standard radiological evaluation led to the consideration of either non-infectious pulmonary diseases (such as, cardiac failure, pulmonary embolism, pulmonary neoplasia or bronchitis) or extra-pulmonary infectious diseases as CAP. Of note, some of these diseases are also associated with increased biomarker values.", "Of note, some of these diseases are also associated with increased biomarker values. This raises concerns about previous evaluations of biomarkers in CAP-suspected patients, which used clinical and standard radiological (chest X-ray) evaluations as the gold standard for CAP diagnosis [15] . The use of biomarkers has been advocated to improve diagnosis and management of patients with lower respiratory tract infections [14] .", "The use of biomarkers has been advocated to improve diagnosis and management of patients with lower respiratory tract infections [14] . However, this issue is still unresolved [24] , with conflicting positions [14, 15, 25, 26] . In our study, while median values of both biomarkers did increase with level of certainty for CAP diagnosis, we were unable to establish discriminating values for PCT.", "In our study, while median values of both biomarkers did increase with level of certainty for CAP diagnosis, we were unable to establish discriminating values for PCT. Recent data suggested that CRP could be of more help in assisting in the diagnosis of lower respiratory tract infections (LRTI) [15, 27, 28] . In our study, although CRP seems more discriminating than PCT, neither the experimental exclusion of extra-pulmonary bacterial infections from the excluded CAP group, nor the exclusion of viral CAP from the definite CAP patients group, made possible the determination of a discriminant cutoff.", "In our study, although CRP seems more discriminating than PCT, neither the experimental exclusion of extra-pulmonary bacterial infections from the excluded CAP group, nor the exclusion of viral CAP from the definite CAP patients group, made possible the determination of a discriminant cutoff. The combination of CRP and PCT was not more discriminating than each biomarker separately. An operational algorithm has been released to assist physicians in prescribing antimicrobial therapy [14, 26, 29] .", "An operational algorithm has been released to assist physicians in prescribing antimicrobial therapy [14, 26, 29] . According to this strategy, a PCT concentration higher than 0.25 μg/L should prompt administration of antibiotics to patients with suspected LRTI. In our study, this value was associated with poor performance.", "In our study, this value was associated with poor performance. Additionally, mean PCT levels remained above this threshold both in excluded CAP patients without infectious disorders and in definite CAP presumably related to virus. Therefore, the gold standard for the diagnosis of CAP may influence the performance and utility of PCT in this setting.", "Therefore, the gold standard for the diagnosis of CAP may influence the performance and utility of PCT in this setting. This study has some limitations. First, the adjudication committee was not blinded to the value of biomarkers measured at bedside in some patients (70 for CRP and 131 for PCT) and its CAP classification could thus have been influenced by these results.", "First, the adjudication committee was not blinded to the value of biomarkers measured at bedside in some patients (70 for CRP and 131 for PCT) and its CAP classification could thus have been influenced by these results. However, the lack of statistically significant differences in the mean CRP and PCT values in the definite CAP cases, whether or not these biomarkers were available for the adjudication committee, argues against a major impact of these results on adjudication committee classification. Second, another critical point is the prescription of antibiotic therapy (34 %) previous to inclusion.", "Second, another critical point is the prescription of antibiotic therapy (34 %) previous to inclusion. We cannot exclude that these previously-treated CAP patients may have altered biomarker performance and reduced the yield of bacterial cultures, although such a population reflects the usual emergency department practice. Third, multiplex PCR was performed on naso-pharyngeal sampling and not on lower respiratory tract samples, which does not allow definite confirmation of the viral origin of CAP.", "Third, multiplex PCR was performed on naso-pharyngeal sampling and not on lower respiratory tract samples, which does not allow definite confirmation of the viral origin of CAP. However, a recent large study on CAP patients which reported a viral etiology of CAP at a comparable rate, did not find upper respiratory tract shedding in a control population without CAP explored during the same year and season [30] . Finally, even if multidetector thoracic CT scan is a better imaging examination than X-ray to explore the chest, only invasive local microbiological samples would have provided a diagnosis with certainty.", "Finally, even if multidetector thoracic CT scan is a better imaging examination than X-ray to explore the chest, only invasive local microbiological samples would have provided a diagnosis with certainty. Given the diversity of the clinical and radiological CAP presentations, CAP diagnosis is often uncertain. In our population of patients treated in the emergency room with clinical symptoms evoking CAP, neither CRP nor PCT cut-off values carried sufficient weight to confirm or refute CAP diagnosis at bedside; this underlines that these biomarkers are telltales of the host inflammatory response to the intrusion of microorganisms independent of the site of infection.", "In our population of patients treated in the emergency room with clinical symptoms evoking CAP, neither CRP nor PCT cut-off values carried sufficient weight to confirm or refute CAP diagnosis at bedside; this underlines that these biomarkers are telltales of the host inflammatory response to the intrusion of microorganisms independent of the site of infection. These results, based on a systematic thoracic CT scan evaluation of CAP-suspected patients, do not argue for the use of CRP and PCT in routine care to diagnose CAP with certainty in patients visiting the ED for suspected CAP." ]

[ "INTRODUCTION: Community-acquired pneumonia (CAP) requires prompt treatment, but its diagnosis is complex. Improvement of bacterial CAP diagnosis by biomarkers has been evaluated using chest X-ray infiltrate as the CAP gold standard, producing conflicting results. We analyzed the diagnostic accuracy of biomarkers in suspected CAP adults visiting emergency departments for whom CAP diagnosis was established by an adjudication committee which founded its judgment on a systematic multidetector thoracic CT scan.", "We analyzed the diagnostic accuracy of biomarkers in suspected CAP adults visiting emergency departments for whom CAP diagnosis was established by an adjudication committee which founded its judgment on a systematic multidetector thoracic CT scan. METHODS: In an ancillary study of a multi-center prospective study evaluating the impact of systematic thoracic CT scan on CAP diagnosis, sensitivity and specificity of C-reactive protein (CRP) and procalcitonin (PCT) were evaluated. Systematic nasopharyngeal multiplex respiratory virus PCR was performed at inclusion.", "Systematic nasopharyngeal multiplex respiratory virus PCR was performed at inclusion. An adjudication committee classified CAP diagnostic probability on a 4-level Likert scale, based on all available data. RESULTS: Two hundred patients with suspected CAP were analyzed.", "RESULTS: Two hundred patients with suspected CAP were analyzed. The adjudication committee classified 98 patients (49.0 %) as definite CAP, 8 (4.0 %) as probable, 23 (11.5 %) as possible and excluded in 71 (35.5 %, including 29 patients with pulmonary infiltrates on chest X-ray). Among patients with radiological pulmonary infiltrate, 23 % were finally classified as excluded.", "Among patients with radiological pulmonary infiltrate, 23 % were finally classified as excluded. Viruses were identified by PCR in 29 % of patients classified as definite. Area under the curve was 0.787 [95 % confidence interval (95 % CI), 0.717 to 0.857] for CRP and 0.655 (95 % CI, 0.570 to 0.739) for PCT to detect definite CAP.", "Area under the curve was 0.787 [95 % confidence interval (95 % CI), 0.717 to 0.857] for CRP and 0.655 (95 % CI, 0.570 to 0.739) for PCT to detect definite CAP. CRP threshold at 50 mg/L resulted in a positive predictive value of 0.76 and a negative predictive value of 0.75. No PCT cut-off resulted in satisfactory positive or negative predictive values.", "No PCT cut-off resulted in satisfactory positive or negative predictive values. CRP and PCT accuracy was not improved by exclusion of the 25 (25.5 %) definite viral CAP cases. CONCLUSIONS: For patients with suspected CAP visiting emergency departments, diagnostic accuracy of CRP and PCT are insufficient to confirm the CAP diagnosis established using a gold standard that includes thoracic CT scan.", "CONCLUSIONS: For patients with suspected CAP visiting emergency departments, diagnostic accuracy of CRP and PCT are insufficient to confirm the CAP diagnosis established using a gold standard that includes thoracic CT scan. Diagnostic accuracy of these biomarkers is also insufficient to distinguish bacterial CAP from viral CAP. TRIAL REGISTRATION: ClinicalTrials.gov registry NCT01574066 (February 7, 2012) ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (.1186/s13054-015-1083-6) contains supplementary material, which is available to authorized users.", "TRIAL REGISTRATION: ClinicalTrials.gov registry NCT01574066 (February 7, 2012) ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (.1186/s13054-015-1083-6) contains supplementary material, which is available to authorized users. Text: Community-acquired pneumonia (CAP) is a frequently seen disease, with high morbidity and mortality, accounting for 600,000 hospitalizations each year. It represents the seventh leading cause of death in the USA [1] .", "It represents the seventh leading cause of death in the USA [1] . CAP prognosis depends on the rapidity of specific treatment, which should ideally be initiated within four hours and no later than eight hours after diagnosis [2, 3] . CAP diagnosis is based on the clustering of non-specific pulmonary and general symptoms [4, 5] , an increase in biomarkers reflecting systemic inflammatory response syndrome (SIRS), and the presence of new parenchymal infiltrates on chest X-ray.", "CAP diagnosis is based on the clustering of non-specific pulmonary and general symptoms [4, 5] , an increase in biomarkers reflecting systemic inflammatory response syndrome (SIRS), and the presence of new parenchymal infiltrates on chest X-ray. However, CAP diagnosis remains uncertain in many cases with alternative diagnoses, such as cardiac failure, acute bronchitis, chronic obstructive pulmonary disease (COPD) exacerbations, pulmonary embolism, neoplasia, and sepsis [6, 7] . Part of the uncertainty of CAP diagnosis may be due to the high rate of chest X-ray misdiagnosis [8, 9] ; over diagnosis of CAP is frequent when infiltrates of noninfectious origin coexist with pulmonary or general symptoms, and the diagnosis of CAP is often ignored when the lung infiltrates are at the limit of visibility or are hidden due to superposition [10] .", "Part of the uncertainty of CAP diagnosis may be due to the high rate of chest X-ray misdiagnosis [8, 9] ; over diagnosis of CAP is frequent when infiltrates of noninfectious origin coexist with pulmonary or general symptoms, and the diagnosis of CAP is often ignored when the lung infiltrates are at the limit of visibility or are hidden due to superposition [10] . We recently published a study in which thoracic CT scan was systematically performed in a population of clinically suspected CAP patients visiting the emergency department for CAP (the ESCAPED study) [11] . We showed that CAP diagnosis based on chest X-ray led to a false CAP diagnosis in many patients: among CAP suspected patients with radiological pulmonary infiltrate, CAP diagnosis was excluded in around 30 % of patients based on CT scan results; on the contrary, among patients without radiological pulmonary infiltrate, one-third had a pulmonary infiltrate on thoracic CT-scan.", "We showed that CAP diagnosis based on chest X-ray led to a false CAP diagnosis in many patients: among CAP suspected patients with radiological pulmonary infiltrate, CAP diagnosis was excluded in around 30 % of patients based on CT scan results; on the contrary, among patients without radiological pulmonary infiltrate, one-third had a pulmonary infiltrate on thoracic CT-scan. We also reported the isolation of viruses in one-third of patients [11, 12] . Several attempts have been made to improve CAP diagnosis based on biomarkers, such as C-reactive protein (CRP) and procalcitonin (PCT); however, there are conflicting data on their reliability [13] [14] [15] [16] [17] .", "Several attempts have been made to improve CAP diagnosis based on biomarkers, such as C-reactive protein (CRP) and procalcitonin (PCT); however, there are conflicting data on their reliability [13] [14] [15] [16] [17] . This could be due to the consideration of CAP diagnosis based on chest X-ray as establishing pulmonary infection. In the present study, we aimed to analyze CRP and PCT values in the population of the ESCAPED study reported above for whom CAP diagnosis was established by an adjudication committee which founded its judgment on all usual available data, systematic multidetector thoracic CT scan performed at inclusion, and results from a day-28 follow-up.", "In the present study, we aimed to analyze CRP and PCT values in the population of the ESCAPED study reported above for whom CAP diagnosis was established by an adjudication committee which founded its judgment on all usual available data, systematic multidetector thoracic CT scan performed at inclusion, and results from a day-28 follow-up. We also analyzed whether the viral etiology of definite CAP based on polymerase chain reaction (PCR) multiplex naso-pharyngeal swab interfered with the accuracy of the biomarkers. Setting ESCAPED was a multicenter, prospective, interventional study, entitled \"Early Thoracic CT-Scan for Community-Acquired Pneumonia at the Emergency Department (ESCAPED)\" [11] , conducted from November 2011 to January 2013, in four emergency departments (EDs) of four tertiary teaching hospitals in Paris, France, designed to measure the impact of thoracic CT scan on clinical decision.", "Setting ESCAPED was a multicenter, prospective, interventional study, entitled \"Early Thoracic CT-Scan for Community-Acquired Pneumonia at the Emergency Department (ESCAPED)\" [11] , conducted from November 2011 to January 2013, in four emergency departments (EDs) of four tertiary teaching hospitals in Paris, France, designed to measure the impact of thoracic CT scan on clinical decision. The study was sponsored and monitored by the Paris public health hospitals, and funded by the French Ministry of Health. The French health authorities (Agence nationale de sécurité des medicaments et produits de santé, ANSM) and the institutional review board for the protection of human subjects approved the study protocol and patient informed consent procedures.", "The French health authorities (Agence nationale de sécurité des medicaments et produits de santé, ANSM) and the institutional review board for the protection of human subjects approved the study protocol and patient informed consent procedures. All enrolled patients provided written informed consent for inclusion. The protocol was registered in the clinicaltrial.gov website under the PACSCAN acronym, the French translation of the English ESCAPED acronym (NCT01574066).", "The protocol was registered in the clinicaltrial.gov website under the PACSCAN acronym, the French translation of the English ESCAPED acronym (NCT01574066). The Ethics Committee of Ile de France (Comité de Protection des Personnes. Paris N°2 011-oct-12749) approved the study protocol.", "Paris N°2 011-oct-12749) approved the study protocol. The primary objective was to compare CRP and PCT values in the four different categories of CAP level of certainty using the day-28 adjudication committee classification. The four categories were: 1) absence of CAP hereafter referred to as excluded CAP diagnosis; 2) possible CAP; 3) probable CAP; and 4) definite CAP.", "The four categories were: 1) absence of CAP hereafter referred to as excluded CAP diagnosis; 2) possible CAP; 3) probable CAP; and 4) definite CAP. The secondary objectives were to assess whether CRP and PCT were associated with CAP diagnosis using sensitivity analyses in three successive subgroups chosen a priori; 1) when specifically considering patients classified as having excluded CAP diagnosis and definite CAP (i.e., the patients for whom the level of certainty was the highest); 2) when patients with excluded CAP diagnosis and diagnosed extra-pulmonary infectious disease (which may increase biomarker values) were not taken into account, in the excluded CAP group; and 3) when patients classified as viral CAP were not taken into account in the definite CAP group, as PCT has been reported to be lower in viral infections as compared to bacterial infections [18] . Consecutive adults ( [19] .", "Consecutive adults ( [19] . Multidetector thoracic CT-scan was performed after chest X-ray, ideally within the four hours following inclusion. Chest X-ray and thoracic CT-scan were performed using a standardized protocol.", "Chest X-ray and thoracic CT-scan were performed using a standardized protocol. The four levels of CAP probability according to CT scan were defined as definite (systematic alveolar condensation, alveolar condensation with peripheral and localized ground glass opacities, bronchiolar focal or multifocal micronodules), probable (peripheral alveolar condensation, retractile systematic alveolar condensation, or diffuse ground glass opacities), possible (pulmonary infarct), or excluded (pulmonary mass, other abnormalities, or normal images). Scan views were recorded on a DVD.", "Scan views were recorded on a DVD. Based on data collected from baseline standardized case report forms, DVD recorded pictures of X-ray and CTscan, and blinded to local interpretations, an adjudication committee consisting of three independent senior experts in infectious diseases, pneumology and radiology retrospectively assigned the probability of CAP diagnosis using the same 4-level Likert scale, with all available data including patients' discharge summary, and follow-up data obtained by assistant investigators who contacted by phone either the patient, relatives or general practitioners at day 28. For this study, the gold standard of CAP was the diagnosis assessed by this adjudication committee.", "For this study, the gold standard of CAP was the diagnosis assessed by this adjudication committee. Alternative diagnoses were established for excluded CAP and classified as non-CAP pulmonary diseases and extra-pulmonary infectious diseases and others. Blood samples were collected at inclusion in sodium heparin-treated tubes, centrifuged, and stored at −40°C until completion of the study.", "Blood samples were collected at inclusion in sodium heparin-treated tubes, centrifuged, and stored at −40°C until completion of the study. CRP and PCT concentrations were measured a posteriori on plasma collection (see Additional file 1 for methodology), except for patients in whom marker dosage was performed by the emergency practitioner on his own initiative. Naso-pharyngeal swabs were collected at enrollment and placed in a Middle Virocult MWE (Sigma®) transport medium.", "Naso-pharyngeal swabs were collected at enrollment and placed in a Middle Virocult MWE (Sigma®) transport medium. Samples were kept at room temperature and sent to the virology laboratory of Bichat -Claude Bernard Hospital (Paris) as soon as possible after collection. The samples were not frozen and thawed.", "The samples were not frozen and thawed. Multiplex PCR (RespiFinder-19 assay (Pathofinder®, Maastricht, Netherlands)) was performed on naso-pharyngeal swabs to detect 15 respiratory viruses -coronavirus 229E, NL63, OC43, human metapneumovirus (hMPV), influenza A, A (H1N1) pdm2009 and B viruses, parainfluenza viruses 1, 2, 3, and 4, respiratory syncytial virus (RSV) A and B, rhinovirus, adenovirus, and 4 intracellular bacteria -Bordetella pertussis, Chlamydophila pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, in one reaction. The multiplex PCR results were not available to the adjudication committee.", "The multiplex PCR results were not available to the adjudication committee. Routine microbiological examinations were also performed at the discretion of the emergency physicians and included blood culture, sputum culture, and antigenuria (see Additional file 1 for methodology). CAP, classified as definite, was considered as being of viral origin when multiplex PCR was positive for at least one of the 15 respiratory viruses and no bacteria were found using PCR and routine bacterial microbiological samples (sputum, blood culture, antigenuria) when performed.", "CAP, classified as definite, was considered as being of viral origin when multiplex PCR was positive for at least one of the 15 respiratory viruses and no bacteria were found using PCR and routine bacterial microbiological samples (sputum, blood culture, antigenuria) when performed. Baseline and follow-up characteristics were described by means and standard deviations (SD) or by median and interquartile range (IQR) for continuous variables normally distributed or with skewed distribution, respectively, and by percentages for categorical variables, for the total study population and for the study groups. We performed chi-square or Fisher exact tests when appropriate for qualitative variables, and the Student or Mann-Whitney tests for continuous variables with skewed distributions to compare baseline patient characteristics and study outcomes between study groups.", "We performed chi-square or Fisher exact tests when appropriate for qualitative variables, and the Student or Mann-Whitney tests for continuous variables with skewed distributions to compare baseline patient characteristics and study outcomes between study groups. The distribution values of the biomarkers were determined in the different populations of patients using boxplots. The performances of CRP and PCT in predicting definite CAP were evaluated by sensitivity analysis (definite CAP vs excluded CAP).", "The performances of CRP and PCT in predicting definite CAP were evaluated by sensitivity analysis (definite CAP vs excluded CAP). CRP was evaluated at several cut-off points of 20 mg/L, 30 mg/L, 50 mg/L, 70 mg/L, and 100 mg/L, values used in previous studies [15, 20, 21] . Several cut-off points for PCT were chosen at the level of 0.10 μg/L [18] , and at the two levels for suspected bacterial infection as stated by the manufacturer, i.e., 0.25 μg/L and 0.50 μg/L.", "Several cut-off points for PCT were chosen at the level of 0.10 μg/L [18] , and at the two levels for suspected bacterial infection as stated by the manufacturer, i.e., 0.25 μg/L and 0.50 μg/L. Sensitivities, specificities, positive predictive values (PPVs), negative predictive values (NPVs), and likelihood ratio were calculated. Receiver operating characteristic (ROC) curves were drawn, area under the curve AUC was computed and optimal cut-off was identified by the maximization of the Youden's index, comparing biomarker values in patients with excluded CAP and definite CAP.", "Receiver operating characteristic (ROC) curves were drawn, area under the curve AUC was computed and optimal cut-off was identified by the maximization of the Youden's index, comparing biomarker values in patients with excluded CAP and definite CAP. From these optimal cut-offs for CRP and PCT, sensitivity analyses were performed combining the CRP and PCT cut-offs. A multivariate logistic regression model was built to identify factors associated with having definite CAP as compared to having an excluded CAP diagnosis.", "A multivariate logistic regression model was built to identify factors associated with having definite CAP as compared to having an excluded CAP diagnosis. We excluded from the excluded CAP diagnosis group, patients with an extra-pulmonary infectious disease. All variables with a p value of < 0.25 in the bivariate analysis were entered into a multivariate logistic regression with a backward stepwise approach; the discrimination was evaluated by the C-index and its 95 % confidence interval (95 % CI) and the calibration was evaluated by the Hosmer Lemeshow goodness-of-fit test.", "All variables with a p value of < 0.25 in the bivariate analysis were entered into a multivariate logistic regression with a backward stepwise approach; the discrimination was evaluated by the C-index and its 95 % confidence interval (95 % CI) and the calibration was evaluated by the Hosmer Lemeshow goodness-of-fit test. All tests were two-sided, and p-values below 0.05 were considered to denote statistical significance. All statistical analyses were performed using SPSS statistical software version 21.0 (SPSS Inc., Chicago, IL, USA).", "All statistical analyses were performed using SPSS statistical software version 21.0 (SPSS Inc., Chicago, IL, USA). Two hundred patients with suspected CAP out of the 319 in the ESCAPED study were included in the present study, for which CRP and PCT assays and nasopharyngeal swab for multiplex PCR were available (Fig. 1) .", "1) . Characteristics of the 200 patients (age, age more than 65, gender, probability of CAP diagnosis by adjudication committee) were not significantly different from those of the 119 other patients of the ESCAPED study and are summarized in Table 1 . CRP and PCT assays were performed based on the emergency practitioner's own initiative in 70 patients for CRP and 131 for PCT, or performed a posteriori on plasma samples of the remaining patients.", "CRP and PCT assays were performed based on the emergency practitioner's own initiative in 70 patients for CRP and 131 for PCT, or performed a posteriori on plasma samples of the remaining patients. Sex ratio was approximately 1. More than half of the patients (54 %) were 65 years of age or older.", "More than half of the patients (54 %) were 65 years of age or older. The \n\nPulmonary infiltrates were seen on chest X-ray in 127 (63.5 %) patients. Thoracic CT-scan excluded a CAP diagnosis in 16.5 % of these 127 patients; on the contrary, thoracic CT-scan revealed a parenchymal infiltrate in 27 % of the 73 patients without infiltrate on chest X-ray.", "Thoracic CT-scan excluded a CAP diagnosis in 16.5 % of these 127 patients; on the contrary, thoracic CT-scan revealed a parenchymal infiltrate in 27 % of the 73 patients without infiltrate on chest X-ray. Based on all available data including multidetector CT scan results (but excluding PCR results), the adjudication \n\nThe CRP and PCT distributions in the 200 patients are presented in Fig. 2 A statistically significant difference between the two groups (excluded CAP vs definite CAP) was demonstrated for several cut-off points for CRP and PCT ( Table 2 ).", "2 A statistically significant difference between the two groups (excluded CAP vs definite CAP) was demonstrated for several cut-off points for CRP and PCT ( Table 2 ). For CRP, the value of 50 mg/L resulted in a PPV of 0.76 and a NPV of 0.75. For PCT, no value resulted in a satisfactory PPV or NPV.", "For PCT, no value resulted in a satisfactory PPV or NPV. For these two biochemical markers, the ability to predict CAP was evaluated by a ROC curve. The AUC was 0.787 (95 % CI 0.717-0.857), optimal cut-off = 45.9 mg/L for CRP (Fig.", "The AUC was 0.787 (95 % CI 0.717-0.857), optimal cut-off = 45.9 mg/L for CRP (Fig. 3 ) and 0.655 (95 % CI 0.570-0.739), optimal cut-off = 0.13 μg/ L for PCT (Fig. 4) .", "4) . Sensitivity analyses for the combination of CRP and PCT, using these optimal cut-offs, resulted in a PPV of 0.74 and a NPV of 0.58. Use of the other PCT cut-offs did not result in better PPV or NPV ( Table 2) .", "Use of the other PCT cut-offs did not result in better PPV or NPV ( Table 2) . The present study is novel as patients prospectively benefited from extensive investigation to determine the diagnosis of CAP in the ED, including both early multidetector thoracic CT-scan and day-28 adjudication committee. This led to the correction of CAP diagnosis previously based on chest X-ray in a high number of patients.", "This led to the correction of CAP diagnosis previously based on chest X-ray in a high number of patients. In these extensively characterized patients, both CRP and PCT lacked operational precision to allow the decisionmaking process to rule out or confirm diagnosis of CAP even in selected subgroups. The clinical characteristics of the patients included in this sub-study are consistent with those in the current literature.", "The clinical characteristics of the patients included in this sub-study are consistent with those in the current literature. As previously reported, patients frequently had a history of respiratory disorders, cancer and congestive heart failure [21, 22] . The design of the ESCAPED study required exclusion of patients within the highest CRB 65 categories, which limited the inclusion of patients older than 65.", "The design of the ESCAPED study required exclusion of patients within the highest CRB 65 categories, which limited the inclusion of patients older than 65. This may explain why the mean age of our patients (64 years) falls within the lower values of those reported elsewhere [19] . Data to identify the microbial agent responsible for the disease were collected by the usual techniques and multiplex PCR.", "Data to identify the microbial agent responsible for the disease were collected by the usual techniques and multiplex PCR. Viral identification using naso-pharyngeal PCR that revealed viral respiratory infection in approximately one-third of cases was concordant with values reported in the literature [23] . Therefore, we believe that our results can be extrapolated to most emergency patients suffering from CAP.", "Therefore, we believe that our results can be extrapolated to most emergency patients suffering from CAP. In the present study, patients were recruited on the basis of initial clinical assessment for the diagnosis of CAP. Therefore, we believe that the characteristics of the patients closely correspond to those that lead practitioners to consider a possible diagnosis of CAP.", "Therefore, we believe that the characteristics of the patients closely correspond to those that lead practitioners to consider a possible diagnosis of CAP. In these patients, the design of our study allowed us to confirm or refute CAP diagnosis with a high level of certainty. Results confirmed the poor predictive value of clinical symptoms (new onset of systemic features and symptoms of an acute lower respiratory tract illness) in identifying CAP patients [21] .", "Results confirmed the poor predictive value of clinical symptoms (new onset of systemic features and symptoms of an acute lower respiratory tract illness) in identifying CAP patients [21] . Indeed, clinical presentation of excluded CAP patients was similar to that of definite CAP patients except for fever and cough that were more frequent in definite CAP patients. Furthermore, the design also revealed that the combination of clinical symptoms and chest X-ray results led to CAP misdiagnosis in a high number of patients, including the 98 whose CAP diagnosis was excluded by the adjudication committee and who would have been considered as possible, probable or definite CAP without the use of the CT scan.", "Furthermore, the design also revealed that the combination of clinical symptoms and chest X-ray results led to CAP misdiagnosis in a high number of patients, including the 98 whose CAP diagnosis was excluded by the adjudication committee and who would have been considered as possible, probable or definite CAP without the use of the CT scan. This low specificity of clinical-standard radiological evaluation led to the consideration of either non-infectious pulmonary diseases (such as, cardiac failure, pulmonary embolism, pulmonary neoplasia or bronchitis) or extra-pulmonary infectious diseases as CAP. Of note, some of these diseases are also associated with increased biomarker values.", "Of note, some of these diseases are also associated with increased biomarker values. This raises concerns about previous evaluations of biomarkers in CAP-suspected patients, which used clinical and standard radiological (chest X-ray) evaluations as the gold standard for CAP diagnosis [15] . The use of biomarkers has been advocated to improve diagnosis and management of patients with lower respiratory tract infections [14] .", "The use of biomarkers has been advocated to improve diagnosis and management of patients with lower respiratory tract infections [14] . However, this issue is still unresolved [24] , with conflicting positions [14, 15, 25, 26] . In our study, while median values of both biomarkers did increase with level of certainty for CAP diagnosis, we were unable to establish discriminating values for PCT.", "In our study, while median values of both biomarkers did increase with level of certainty for CAP diagnosis, we were unable to establish discriminating values for PCT. Recent data suggested that CRP could be of more help in assisting in the diagnosis of lower respiratory tract infections (LRTI) [15, 27, 28] . In our study, although CRP seems more discriminating than PCT, neither the experimental exclusion of extra-pulmonary bacterial infections from the excluded CAP group, nor the exclusion of viral CAP from the definite CAP patients group, made possible the determination of a discriminant cutoff.", "In our study, although CRP seems more discriminating than PCT, neither the experimental exclusion of extra-pulmonary bacterial infections from the excluded CAP group, nor the exclusion of viral CAP from the definite CAP patients group, made possible the determination of a discriminant cutoff. The combination of CRP and PCT was not more discriminating than each biomarker separately. An operational algorithm has been released to assist physicians in prescribing antimicrobial therapy [14, 26, 29] .", "An operational algorithm has been released to assist physicians in prescribing antimicrobial therapy [14, 26, 29] . According to this strategy, a PCT concentration higher than 0.25 μg/L should prompt administration of antibiotics to patients with suspected LRTI. In our study, this value was associated with poor performance.", "In our study, this value was associated with poor performance. Additionally, mean PCT levels remained above this threshold both in excluded CAP patients without infectious disorders and in definite CAP presumably related to virus. Therefore, the gold standard for the diagnosis of CAP may influence the performance and utility of PCT in this setting.", "Therefore, the gold standard for the diagnosis of CAP may influence the performance and utility of PCT in this setting. This study has some limitations. First, the adjudication committee was not blinded to the value of biomarkers measured at bedside in some patients (70 for CRP and 131 for PCT) and its CAP classification could thus have been influenced by these results.", "First, the adjudication committee was not blinded to the value of biomarkers measured at bedside in some patients (70 for CRP and 131 for PCT) and its CAP classification could thus have been influenced by these results. However, the lack of statistically significant differences in the mean CRP and PCT values in the definite CAP cases, whether or not these biomarkers were available for the adjudication committee, argues against a major impact of these results on adjudication committee classification. Second, another critical point is the prescription of antibiotic therapy (34 %) previous to inclusion.", "Second, another critical point is the prescription of antibiotic therapy (34 %) previous to inclusion. We cannot exclude that these previously-treated CAP patients may have altered biomarker performance and reduced the yield of bacterial cultures, although such a population reflects the usual emergency department practice. Third, multiplex PCR was performed on naso-pharyngeal sampling and not on lower respiratory tract samples, which does not allow definite confirmation of the viral origin of CAP.", "Third, multiplex PCR was performed on naso-pharyngeal sampling and not on lower respiratory tract samples, which does not allow definite confirmation of the viral origin of CAP. However, a recent large study on CAP patients which reported a viral etiology of CAP at a comparable rate, did not find upper respiratory tract shedding in a control population without CAP explored during the same year and season [30] . Finally, even if multidetector thoracic CT scan is a better imaging examination than X-ray to explore the chest, only invasive local microbiological samples would have provided a diagnosis with certainty.", "Finally, even if multidetector thoracic CT scan is a better imaging examination than X-ray to explore the chest, only invasive local microbiological samples would have provided a diagnosis with certainty. Given the diversity of the clinical and radiological CAP presentations, CAP diagnosis is often uncertain. In our population of patients treated in the emergency room with clinical symptoms evoking CAP, neither CRP nor PCT cut-off values carried sufficient weight to confirm or refute CAP diagnosis at bedside; this underlines that these biomarkers are telltales of the host inflammatory response to the intrusion of microorganisms independent of the site of infection.", "In our population of patients treated in the emergency room with clinical symptoms evoking CAP, neither CRP nor PCT cut-off values carried sufficient weight to confirm or refute CAP diagnosis at bedside; this underlines that these biomarkers are telltales of the host inflammatory response to the intrusion of microorganisms independent of the site of infection. These results, based on a systematic thoracic CT scan evaluation of CAP-suspected patients, do not argue for the use of CRP and PCT in routine care to diagnose CAP with certainty in patients visiting the ED for suspected CAP." ]

[ "INTRODUCTION: Community-acquired pneumonia (CAP) requires prompt treatment, but its diagnosis is complex. Improvement of bacterial CAP diagnosis by biomarkers has been evaluated using chest X-ray infiltrate as the CAP gold standard, producing conflicting results. We analyzed the diagnostic accuracy of biomarkers in suspected CAP adults visiting emergency departments for whom CAP diagnosis was established by an adjudication committee which founded its judgment on a systematic multidetector thoracic CT scan.", "We analyzed the diagnostic accuracy of biomarkers in suspected CAP adults visiting emergency departments for whom CAP diagnosis was established by an adjudication committee which founded its judgment on a systematic multidetector thoracic CT scan. METHODS: In an ancillary study of a multi-center prospective study evaluating the impact of systematic thoracic CT scan on CAP diagnosis, sensitivity and specificity of C-reactive protein (CRP) and procalcitonin (PCT) were evaluated. Systematic nasopharyngeal multiplex respiratory virus PCR was performed at inclusion.", "Systematic nasopharyngeal multiplex respiratory virus PCR was performed at inclusion. An adjudication committee classified CAP diagnostic probability on a 4-level Likert scale, based on all available data. RESULTS: Two hundred patients with suspected CAP were analyzed.", "RESULTS: Two hundred patients with suspected CAP were analyzed. The adjudication committee classified 98 patients (49.0 %) as definite CAP, 8 (4.0 %) as probable, 23 (11.5 %) as possible and excluded in 71 (35.5 %, including 29 patients with pulmonary infiltrates on chest X-ray). Among patients with radiological pulmonary infiltrate, 23 % were finally classified as excluded.", "Among patients with radiological pulmonary infiltrate, 23 % were finally classified as excluded. Viruses were identified by PCR in 29 % of patients classified as definite. Area under the curve was 0.787 [95 % confidence interval (95 % CI), 0.717 to 0.857] for CRP and 0.655 (95 % CI, 0.570 to 0.739) for PCT to detect definite CAP.", "Area under the curve was 0.787 [95 % confidence interval (95 % CI), 0.717 to 0.857] for CRP and 0.655 (95 % CI, 0.570 to 0.739) for PCT to detect definite CAP. CRP threshold at 50 mg/L resulted in a positive predictive value of 0.76 and a negative predictive value of 0.75. No PCT cut-off resulted in satisfactory positive or negative predictive values.", "No PCT cut-off resulted in satisfactory positive or negative predictive values. CRP and PCT accuracy was not improved by exclusion of the 25 (25.5 %) definite viral CAP cases. CONCLUSIONS: For patients with suspected CAP visiting emergency departments, diagnostic accuracy of CRP and PCT are insufficient to confirm the CAP diagnosis established using a gold standard that includes thoracic CT scan.", "CONCLUSIONS: For patients with suspected CAP visiting emergency departments, diagnostic accuracy of CRP and PCT are insufficient to confirm the CAP diagnosis established using a gold standard that includes thoracic CT scan. Diagnostic accuracy of these biomarkers is also insufficient to distinguish bacterial CAP from viral CAP. TRIAL REGISTRATION: ClinicalTrials.gov registry NCT01574066 (February 7, 2012) ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (.1186/s13054-015-1083-6) contains supplementary material, which is available to authorized users.", "TRIAL REGISTRATION: ClinicalTrials.gov registry NCT01574066 (February 7, 2012) ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (.1186/s13054-015-1083-6) contains supplementary material, which is available to authorized users. Text: Community-acquired pneumonia (CAP) is a frequently seen disease, with high morbidity and mortality, accounting for 600,000 hospitalizations each year. It represents the seventh leading cause of death in the USA [1] .", "It represents the seventh leading cause of death in the USA [1] . CAP prognosis depends on the rapidity of specific treatment, which should ideally be initiated within four hours and no later than eight hours after diagnosis [2, 3] . CAP diagnosis is based on the clustering of non-specific pulmonary and general symptoms [4, 5] , an increase in biomarkers reflecting systemic inflammatory response syndrome (SIRS), and the presence of new parenchymal infiltrates on chest X-ray.", "CAP diagnosis is based on the clustering of non-specific pulmonary and general symptoms [4, 5] , an increase in biomarkers reflecting systemic inflammatory response syndrome (SIRS), and the presence of new parenchymal infiltrates on chest X-ray. However, CAP diagnosis remains uncertain in many cases with alternative diagnoses, such as cardiac failure, acute bronchitis, chronic obstructive pulmonary disease (COPD) exacerbations, pulmonary embolism, neoplasia, and sepsis [6, 7] . Part of the uncertainty of CAP diagnosis may be due to the high rate of chest X-ray misdiagnosis [8, 9] ; over diagnosis of CAP is frequent when infiltrates of noninfectious origin coexist with pulmonary or general symptoms, and the diagnosis of CAP is often ignored when the lung infiltrates are at the limit of visibility or are hidden due to superposition [10] .", "Part of the uncertainty of CAP diagnosis may be due to the high rate of chest X-ray misdiagnosis [8, 9] ; over diagnosis of CAP is frequent when infiltrates of noninfectious origin coexist with pulmonary or general symptoms, and the diagnosis of CAP is often ignored when the lung infiltrates are at the limit of visibility or are hidden due to superposition [10] . We recently published a study in which thoracic CT scan was systematically performed in a population of clinically suspected CAP patients visiting the emergency department for CAP (the ESCAPED study) [11] . We showed that CAP diagnosis based on chest X-ray led to a false CAP diagnosis in many patients: among CAP suspected patients with radiological pulmonary infiltrate, CAP diagnosis was excluded in around 30 % of patients based on CT scan results; on the contrary, among patients without radiological pulmonary infiltrate, one-third had a pulmonary infiltrate on thoracic CT-scan.", "We showed that CAP diagnosis based on chest X-ray led to a false CAP diagnosis in many patients: among CAP suspected patients with radiological pulmonary infiltrate, CAP diagnosis was excluded in around 30 % of patients based on CT scan results; on the contrary, among patients without radiological pulmonary infiltrate, one-third had a pulmonary infiltrate on thoracic CT-scan. We also reported the isolation of viruses in one-third of patients [11, 12] . Several attempts have been made to improve CAP diagnosis based on biomarkers, such as C-reactive protein (CRP) and procalcitonin (PCT); however, there are conflicting data on their reliability [13] [14] [15] [16] [17] .", "Several attempts have been made to improve CAP diagnosis based on biomarkers, such as C-reactive protein (CRP) and procalcitonin (PCT); however, there are conflicting data on their reliability [13] [14] [15] [16] [17] . This could be due to the consideration of CAP diagnosis based on chest X-ray as establishing pulmonary infection. In the present study, we aimed to analyze CRP and PCT values in the population of the ESCAPED study reported above for whom CAP diagnosis was established by an adjudication committee which founded its judgment on all usual available data, systematic multidetector thoracic CT scan performed at inclusion, and results from a day-28 follow-up.", "In the present study, we aimed to analyze CRP and PCT values in the population of the ESCAPED study reported above for whom CAP diagnosis was established by an adjudication committee which founded its judgment on all usual available data, systematic multidetector thoracic CT scan performed at inclusion, and results from a day-28 follow-up. We also analyzed whether the viral etiology of definite CAP based on polymerase chain reaction (PCR) multiplex naso-pharyngeal swab interfered with the accuracy of the biomarkers. Setting ESCAPED was a multicenter, prospective, interventional study, entitled \"Early Thoracic CT-Scan for Community-Acquired Pneumonia at the Emergency Department (ESCAPED)\" [11] , conducted from November 2011 to January 2013, in four emergency departments (EDs) of four tertiary teaching hospitals in Paris, France, designed to measure the impact of thoracic CT scan on clinical decision.", "Setting ESCAPED was a multicenter, prospective, interventional study, entitled \"Early Thoracic CT-Scan for Community-Acquired Pneumonia at the Emergency Department (ESCAPED)\" [11] , conducted from November 2011 to January 2013, in four emergency departments (EDs) of four tertiary teaching hospitals in Paris, France, designed to measure the impact of thoracic CT scan on clinical decision. The study was sponsored and monitored by the Paris public health hospitals, and funded by the French Ministry of Health. The French health authorities (Agence nationale de sécurité des medicaments et produits de santé, ANSM) and the institutional review board for the protection of human subjects approved the study protocol and patient informed consent procedures.", "The French health authorities (Agence nationale de sécurité des medicaments et produits de santé, ANSM) and the institutional review board for the protection of human subjects approved the study protocol and patient informed consent procedures. All enrolled patients provided written informed consent for inclusion. The protocol was registered in the clinicaltrial.gov website under the PACSCAN acronym, the French translation of the English ESCAPED acronym (NCT01574066).", "The protocol was registered in the clinicaltrial.gov website under the PACSCAN acronym, the French translation of the English ESCAPED acronym (NCT01574066). The Ethics Committee of Ile de France (Comité de Protection des Personnes. Paris N°2 011-oct-12749) approved the study protocol.", "Paris N°2 011-oct-12749) approved the study protocol. The primary objective was to compare CRP and PCT values in the four different categories of CAP level of certainty using the day-28 adjudication committee classification. The four categories were: 1) absence of CAP hereafter referred to as excluded CAP diagnosis; 2) possible CAP; 3) probable CAP; and 4) definite CAP.", "The four categories were: 1) absence of CAP hereafter referred to as excluded CAP diagnosis; 2) possible CAP; 3) probable CAP; and 4) definite CAP. The secondary objectives were to assess whether CRP and PCT were associated with CAP diagnosis using sensitivity analyses in three successive subgroups chosen a priori; 1) when specifically considering patients classified as having excluded CAP diagnosis and definite CAP (i.e., the patients for whom the level of certainty was the highest); 2) when patients with excluded CAP diagnosis and diagnosed extra-pulmonary infectious disease (which may increase biomarker values) were not taken into account, in the excluded CAP group; and 3) when patients classified as viral CAP were not taken into account in the definite CAP group, as PCT has been reported to be lower in viral infections as compared to bacterial infections [18] . Consecutive adults ( [19] .", "Consecutive adults ( [19] . Multidetector thoracic CT-scan was performed after chest X-ray, ideally within the four hours following inclusion. Chest X-ray and thoracic CT-scan were performed using a standardized protocol.", "Chest X-ray and thoracic CT-scan were performed using a standardized protocol. The four levels of CAP probability according to CT scan were defined as definite (systematic alveolar condensation, alveolar condensation with peripheral and localized ground glass opacities, bronchiolar focal or multifocal micronodules), probable (peripheral alveolar condensation, retractile systematic alveolar condensation, or diffuse ground glass opacities), possible (pulmonary infarct), or excluded (pulmonary mass, other abnormalities, or normal images). Scan views were recorded on a DVD.", "Scan views were recorded on a DVD. Based on data collected from baseline standardized case report forms, DVD recorded pictures of X-ray and CTscan, and blinded to local interpretations, an adjudication committee consisting of three independent senior experts in infectious diseases, pneumology and radiology retrospectively assigned the probability of CAP diagnosis using the same 4-level Likert scale, with all available data including patients' discharge summary, and follow-up data obtained by assistant investigators who contacted by phone either the patient, relatives or general practitioners at day 28. For this study, the gold standard of CAP was the diagnosis assessed by this adjudication committee.", "For this study, the gold standard of CAP was the diagnosis assessed by this adjudication committee. Alternative diagnoses were established for excluded CAP and classified as non-CAP pulmonary diseases and extra-pulmonary infectious diseases and others. Blood samples were collected at inclusion in sodium heparin-treated tubes, centrifuged, and stored at −40°C until completion of the study.", "Blood samples were collected at inclusion in sodium heparin-treated tubes, centrifuged, and stored at −40°C until completion of the study. CRP and PCT concentrations were measured a posteriori on plasma collection (see Additional file 1 for methodology), except for patients in whom marker dosage was performed by the emergency practitioner on his own initiative. Naso-pharyngeal swabs were collected at enrollment and placed in a Middle Virocult MWE (Sigma®) transport medium.", "Naso-pharyngeal swabs were collected at enrollment and placed in a Middle Virocult MWE (Sigma®) transport medium. Samples were kept at room temperature and sent to the virology laboratory of Bichat -Claude Bernard Hospital (Paris) as soon as possible after collection. The samples were not frozen and thawed.", "The samples were not frozen and thawed. Multiplex PCR (RespiFinder-19 assay (Pathofinder®, Maastricht, Netherlands)) was performed on naso-pharyngeal swabs to detect 15 respiratory viruses -coronavirus 229E, NL63, OC43, human metapneumovirus (hMPV), influenza A, A (H1N1) pdm2009 and B viruses, parainfluenza viruses 1, 2, 3, and 4, respiratory syncytial virus (RSV) A and B, rhinovirus, adenovirus, and 4 intracellular bacteria -Bordetella pertussis, Chlamydophila pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, in one reaction. The multiplex PCR results were not available to the adjudication committee.", "The multiplex PCR results were not available to the adjudication committee. Routine microbiological examinations were also performed at the discretion of the emergency physicians and included blood culture, sputum culture, and antigenuria (see Additional file 1 for methodology). CAP, classified as definite, was considered as being of viral origin when multiplex PCR was positive for at least one of the 15 respiratory viruses and no bacteria were found using PCR and routine bacterial microbiological samples (sputum, blood culture, antigenuria) when performed.", "CAP, classified as definite, was considered as being of viral origin when multiplex PCR was positive for at least one of the 15 respiratory viruses and no bacteria were found using PCR and routine bacterial microbiological samples (sputum, blood culture, antigenuria) when performed. Baseline and follow-up characteristics were described by means and standard deviations (SD) or by median and interquartile range (IQR) for continuous variables normally distributed or with skewed distribution, respectively, and by percentages for categorical variables, for the total study population and for the study groups. We performed chi-square or Fisher exact tests when appropriate for qualitative variables, and the Student or Mann-Whitney tests for continuous variables with skewed distributions to compare baseline patient characteristics and study outcomes between study groups.", "We performed chi-square or Fisher exact tests when appropriate for qualitative variables, and the Student or Mann-Whitney tests for continuous variables with skewed distributions to compare baseline patient characteristics and study outcomes between study groups. The distribution values of the biomarkers were determined in the different populations of patients using boxplots. The performances of CRP and PCT in predicting definite CAP were evaluated by sensitivity analysis (definite CAP vs excluded CAP).", "The performances of CRP and PCT in predicting definite CAP were evaluated by sensitivity analysis (definite CAP vs excluded CAP). CRP was evaluated at several cut-off points of 20 mg/L, 30 mg/L, 50 mg/L, 70 mg/L, and 100 mg/L, values used in previous studies [15, 20, 21] . Several cut-off points for PCT were chosen at the level of 0.10 μg/L [18] , and at the two levels for suspected bacterial infection as stated by the manufacturer, i.e., 0.25 μg/L and 0.50 μg/L.", "Several cut-off points for PCT were chosen at the level of 0.10 μg/L [18] , and at the two levels for suspected bacterial infection as stated by the manufacturer, i.e., 0.25 μg/L and 0.50 μg/L. Sensitivities, specificities, positive predictive values (PPVs), negative predictive values (NPVs), and likelihood ratio were calculated. Receiver operating characteristic (ROC) curves were drawn, area under the curve AUC was computed and optimal cut-off was identified by the maximization of the Youden's index, comparing biomarker values in patients with excluded CAP and definite CAP.", "Receiver operating characteristic (ROC) curves were drawn, area under the curve AUC was computed and optimal cut-off was identified by the maximization of the Youden's index, comparing biomarker values in patients with excluded CAP and definite CAP. From these optimal cut-offs for CRP and PCT, sensitivity analyses were performed combining the CRP and PCT cut-offs. A multivariate logistic regression model was built to identify factors associated with having definite CAP as compared to having an excluded CAP diagnosis.", "A multivariate logistic regression model was built to identify factors associated with having definite CAP as compared to having an excluded CAP diagnosis. We excluded from the excluded CAP diagnosis group, patients with an extra-pulmonary infectious disease. All variables with a p value of < 0.25 in the bivariate analysis were entered into a multivariate logistic regression with a backward stepwise approach; the discrimination was evaluated by the C-index and its 95 % confidence interval (95 % CI) and the calibration was evaluated by the Hosmer Lemeshow goodness-of-fit test.", "All variables with a p value of < 0.25 in the bivariate analysis were entered into a multivariate logistic regression with a backward stepwise approach; the discrimination was evaluated by the C-index and its 95 % confidence interval (95 % CI) and the calibration was evaluated by the Hosmer Lemeshow goodness-of-fit test. All tests were two-sided, and p-values below 0.05 were considered to denote statistical significance. All statistical analyses were performed using SPSS statistical software version 21.0 (SPSS Inc., Chicago, IL, USA).", "All statistical analyses were performed using SPSS statistical software version 21.0 (SPSS Inc., Chicago, IL, USA). Two hundred patients with suspected CAP out of the 319 in the ESCAPED study were included in the present study, for which CRP and PCT assays and nasopharyngeal swab for multiplex PCR were available (Fig. 1) .", "1) . Characteristics of the 200 patients (age, age more than 65, gender, probability of CAP diagnosis by adjudication committee) were not significantly different from those of the 119 other patients of the ESCAPED study and are summarized in Table 1 . CRP and PCT assays were performed based on the emergency practitioner's own initiative in 70 patients for CRP and 131 for PCT, or performed a posteriori on plasma samples of the remaining patients.", "CRP and PCT assays were performed based on the emergency practitioner's own initiative in 70 patients for CRP and 131 for PCT, or performed a posteriori on plasma samples of the remaining patients. Sex ratio was approximately 1. More than half of the patients (54 %) were 65 years of age or older.", "More than half of the patients (54 %) were 65 years of age or older. The \n\nPulmonary infiltrates were seen on chest X-ray in 127 (63.5 %) patients. Thoracic CT-scan excluded a CAP diagnosis in 16.5 % of these 127 patients; on the contrary, thoracic CT-scan revealed a parenchymal infiltrate in 27 % of the 73 patients without infiltrate on chest X-ray.", "Thoracic CT-scan excluded a CAP diagnosis in 16.5 % of these 127 patients; on the contrary, thoracic CT-scan revealed a parenchymal infiltrate in 27 % of the 73 patients without infiltrate on chest X-ray. Based on all available data including multidetector CT scan results (but excluding PCR results), the adjudication \n\nThe CRP and PCT distributions in the 200 patients are presented in Fig. 2 A statistically significant difference between the two groups (excluded CAP vs definite CAP) was demonstrated for several cut-off points for CRP and PCT ( Table 2 ).", "2 A statistically significant difference between the two groups (excluded CAP vs definite CAP) was demonstrated for several cut-off points for CRP and PCT ( Table 2 ). For CRP, the value of 50 mg/L resulted in a PPV of 0.76 and a NPV of 0.75. For PCT, no value resulted in a satisfactory PPV or NPV.", "For PCT, no value resulted in a satisfactory PPV or NPV. For these two biochemical markers, the ability to predict CAP was evaluated by a ROC curve. The AUC was 0.787 (95 % CI 0.717-0.857), optimal cut-off = 45.9 mg/L for CRP (Fig.", "The AUC was 0.787 (95 % CI 0.717-0.857), optimal cut-off = 45.9 mg/L for CRP (Fig. 3 ) and 0.655 (95 % CI 0.570-0.739), optimal cut-off = 0.13 μg/ L for PCT (Fig. 4) .", "4) . Sensitivity analyses for the combination of CRP and PCT, using these optimal cut-offs, resulted in a PPV of 0.74 and a NPV of 0.58. Use of the other PCT cut-offs did not result in better PPV or NPV ( Table 2) .", "Use of the other PCT cut-offs did not result in better PPV or NPV ( Table 2) . The present study is novel as patients prospectively benefited from extensive investigation to determine the diagnosis of CAP in the ED, including both early multidetector thoracic CT-scan and day-28 adjudication committee. This led to the correction of CAP diagnosis previously based on chest X-ray in a high number of patients.", "This led to the correction of CAP diagnosis previously based on chest X-ray in a high number of patients. In these extensively characterized patients, both CRP and PCT lacked operational precision to allow the decisionmaking process to rule out or confirm diagnosis of CAP even in selected subgroups. The clinical characteristics of the patients included in this sub-study are consistent with those in the current literature.", "The clinical characteristics of the patients included in this sub-study are consistent with those in the current literature. As previously reported, patients frequently had a history of respiratory disorders, cancer and congestive heart failure [21, 22] . The design of the ESCAPED study required exclusion of patients within the highest CRB 65 categories, which limited the inclusion of patients older than 65.", "The design of the ESCAPED study required exclusion of patients within the highest CRB 65 categories, which limited the inclusion of patients older than 65. This may explain why the mean age of our patients (64 years) falls within the lower values of those reported elsewhere [19] . Data to identify the microbial agent responsible for the disease were collected by the usual techniques and multiplex PCR.", "Data to identify the microbial agent responsible for the disease were collected by the usual techniques and multiplex PCR. Viral identification using naso-pharyngeal PCR that revealed viral respiratory infection in approximately one-third of cases was concordant with values reported in the literature [23] . Therefore, we believe that our results can be extrapolated to most emergency patients suffering from CAP.", "Therefore, we believe that our results can be extrapolated to most emergency patients suffering from CAP. In the present study, patients were recruited on the basis of initial clinical assessment for the diagnosis of CAP. Therefore, we believe that the characteristics of the patients closely correspond to those that lead practitioners to consider a possible diagnosis of CAP.", "Therefore, we believe that the characteristics of the patients closely correspond to those that lead practitioners to consider a possible diagnosis of CAP. In these patients, the design of our study allowed us to confirm or refute CAP diagnosis with a high level of certainty. Results confirmed the poor predictive value of clinical symptoms (new onset of systemic features and symptoms of an acute lower respiratory tract illness) in identifying CAP patients [21] .", "Results confirmed the poor predictive value of clinical symptoms (new onset of systemic features and symptoms of an acute lower respiratory tract illness) in identifying CAP patients [21] . Indeed, clinical presentation of excluded CAP patients was similar to that of definite CAP patients except for fever and cough that were more frequent in definite CAP patients. Furthermore, the design also revealed that the combination of clinical symptoms and chest X-ray results led to CAP misdiagnosis in a high number of patients, including the 98 whose CAP diagnosis was excluded by the adjudication committee and who would have been considered as possible, probable or definite CAP without the use of the CT scan.", "Furthermore, the design also revealed that the combination of clinical symptoms and chest X-ray results led to CAP misdiagnosis in a high number of patients, including the 98 whose CAP diagnosis was excluded by the adjudication committee and who would have been considered as possible, probable or definite CAP without the use of the CT scan. This low specificity of clinical-standard radiological evaluation led to the consideration of either non-infectious pulmonary diseases (such as, cardiac failure, pulmonary embolism, pulmonary neoplasia or bronchitis) or extra-pulmonary infectious diseases as CAP. Of note, some of these diseases are also associated with increased biomarker values.", "Of note, some of these diseases are also associated with increased biomarker values. This raises concerns about previous evaluations of biomarkers in CAP-suspected patients, which used clinical and standard radiological (chest X-ray) evaluations as the gold standard for CAP diagnosis [15] . The use of biomarkers has been advocated to improve diagnosis and management of patients with lower respiratory tract infections [14] .", "The use of biomarkers has been advocated to improve diagnosis and management of patients with lower respiratory tract infections [14] . However, this issue is still unresolved [24] , with conflicting positions [14, 15, 25, 26] . In our study, while median values of both biomarkers did increase with level of certainty for CAP diagnosis, we were unable to establish discriminating values for PCT.", "In our study, while median values of both biomarkers did increase with level of certainty for CAP diagnosis, we were unable to establish discriminating values for PCT. Recent data suggested that CRP could be of more help in assisting in the diagnosis of lower respiratory tract infections (LRTI) [15, 27, 28] . In our study, although CRP seems more discriminating than PCT, neither the experimental exclusion of extra-pulmonary bacterial infections from the excluded CAP group, nor the exclusion of viral CAP from the definite CAP patients group, made possible the determination of a discriminant cutoff.", "In our study, although CRP seems more discriminating than PCT, neither the experimental exclusion of extra-pulmonary bacterial infections from the excluded CAP group, nor the exclusion of viral CAP from the definite CAP patients group, made possible the determination of a discriminant cutoff. The combination of CRP and PCT was not more discriminating than each biomarker separately. An operational algorithm has been released to assist physicians in prescribing antimicrobial therapy [14, 26, 29] .", "An operational algorithm has been released to assist physicians in prescribing antimicrobial therapy [14, 26, 29] . According to this strategy, a PCT concentration higher than 0.25 μg/L should prompt administration of antibiotics to patients with suspected LRTI. In our study, this value was associated with poor performance.", "In our study, this value was associated with poor performance. Additionally, mean PCT levels remained above this threshold both in excluded CAP patients without infectious disorders and in definite CAP presumably related to virus. Therefore, the gold standard for the diagnosis of CAP may influence the performance and utility of PCT in this setting.", "Therefore, the gold standard for the diagnosis of CAP may influence the performance and utility of PCT in this setting. This study has some limitations. First, the adjudication committee was not blinded to the value of biomarkers measured at bedside in some patients (70 for CRP and 131 for PCT) and its CAP classification could thus have been influenced by these results.", "First, the adjudication committee was not blinded to the value of biomarkers measured at bedside in some patients (70 for CRP and 131 for PCT) and its CAP classification could thus have been influenced by these results. However, the lack of statistically significant differences in the mean CRP and PCT values in the definite CAP cases, whether or not these biomarkers were available for the adjudication committee, argues against a major impact of these results on adjudication committee classification. Second, another critical point is the prescription of antibiotic therapy (34 %) previous to inclusion.", "Second, another critical point is the prescription of antibiotic therapy (34 %) previous to inclusion. We cannot exclude that these previously-treated CAP patients may have altered biomarker performance and reduced the yield of bacterial cultures, although such a population reflects the usual emergency department practice. Third, multiplex PCR was performed on naso-pharyngeal sampling and not on lower respiratory tract samples, which does not allow definite confirmation of the viral origin of CAP.", "Third, multiplex PCR was performed on naso-pharyngeal sampling and not on lower respiratory tract samples, which does not allow definite confirmation of the viral origin of CAP. However, a recent large study on CAP patients which reported a viral etiology of CAP at a comparable rate, did not find upper respiratory tract shedding in a control population without CAP explored during the same year and season [30] . Finally, even if multidetector thoracic CT scan is a better imaging examination than X-ray to explore the chest, only invasive local microbiological samples would have provided a diagnosis with certainty.", "Finally, even if multidetector thoracic CT scan is a better imaging examination than X-ray to explore the chest, only invasive local microbiological samples would have provided a diagnosis with certainty. Given the diversity of the clinical and radiological CAP presentations, CAP diagnosis is often uncertain. In our population of patients treated in the emergency room with clinical symptoms evoking CAP, neither CRP nor PCT cut-off values carried sufficient weight to confirm or refute CAP diagnosis at bedside; this underlines that these biomarkers are telltales of the host inflammatory response to the intrusion of microorganisms independent of the site of infection.", "In our population of patients treated in the emergency room with clinical symptoms evoking CAP, neither CRP nor PCT cut-off values carried sufficient weight to confirm or refute CAP diagnosis at bedside; this underlines that these biomarkers are telltales of the host inflammatory response to the intrusion of microorganisms independent of the site of infection. These results, based on a systematic thoracic CT scan evaluation of CAP-suspected patients, do not argue for the use of CRP and PCT in routine care to diagnose CAP with certainty in patients visiting the ED for suspected CAP." ]

[ "BACKGROUND: Influenza-like illness (ILI) may be caused by a variety of pathogens. Clinical observations are of little help to recognise myxovirus infection and implement appropriate prevention measures. The limited use of molecular tools underestimates the role of other common pathogens.", "The limited use of molecular tools underestimates the role of other common pathogens. OBJECTIVES: During the early weeks of the 2009–2010 flu pandemic, a clinical and virological survey was conducted in adult and paediatric patients with ILI referred to two French University hospitals in Paris and Tours. Aims were to investigate the different pathogens involved in ILI and describe the associated symptoms.", "Aims were to investigate the different pathogens involved in ILI and describe the associated symptoms. METHODS: H1N1v pandemic influenza diagnosis was performed with real time RT-PCR assay. Other viral aetiologies were investigated by the molecular multiplex assay RespiFinder19®. Clinical data were collected prospectively by physicians using a standard questionnaire.", "Clinical data were collected prospectively by physicians using a standard questionnaire. RESULTS: From week 35 to 44, endonasal swabs were collected in 413 patients. Overall, 68 samples (16.5%) were positive for H1N1v. In 13 of them, other respiratory pathogens were also detected.", "In 13 of them, other respiratory pathogens were also detected. Among H1N1v negative samples, 213 (61.9%) were positive for various respiratory agents, 190 in single infections and 23 in mixed infections. The most prevalent viruses in H1N1v negative single infections were rhinovirus (62.6%), followed by parainfluenza viruses (24.2%) and adenovirus (5.3%).", "The most prevalent viruses in H1N1v negative single infections were rhinovirus (62.6%), followed by parainfluenza viruses (24.2%) and adenovirus (5.3%). 70.6% of H1N1v cases were identified in patients under 40 years and none after 65 years. There was no difference between clinical symptoms observed in patients infected with H1N1v or with other pathogens.", "There was no difference between clinical symptoms observed in patients infected with H1N1v or with other pathogens. CONCLUSION: Our results highlight the high frequency of non-influenza viruses involved in ILI during the pre-epidemic period of a flu alert and the lack of specific clinical signs associated with influenza infections. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management.", "Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management. Text: In order to monitor the spread of influenza and alert health handlers, several epidemiological tools have been developed. In France, a network of 1300 general practitioners, ''Réseau Sentinelles'', working throughout the country, provides real-time clinical data used to evaluate regional and national influenza spreading [1, 2] .", "In France, a network of 1300 general practitioners, ''Réseau Sentinelles'', working throughout the country, provides real-time clinical data used to evaluate regional and national influenza spreading [1, 2] . The criteria used by this network to define clinical influenza-like illness (ILI) are the occurrence of a sudden fever above 39uC with myalgia and respiratory signs. In general no formal viral diagnosis is carried out.", "In general no formal viral diagnosis is carried out. The Groupes Régionaux d'Observation de la Grippe (GROG) is a second French network that surveys the emergence and the spread of the influenza viruses [3, 4] . This network is based on clinical surveillance of acute respiratory infections and laboratory analysis of nasal specimens collected from adults and children by volunteer general practitioners and pediatricians.", "This network is based on clinical surveillance of acute respiratory infections and laboratory analysis of nasal specimens collected from adults and children by volunteer general practitioners and pediatricians. According to the sentinel network's criteria, French health authorities proclaimed that flu epidemic level was reached during the second week of September 2009 (week 37) [5, 6] . On the contrary, data provided by the GROG showed only sporadic H1N1v activity until the last week of October (week 44) [6, 7] .", "On the contrary, data provided by the GROG showed only sporadic H1N1v activity until the last week of October (week 44) [6, 7] . Thus, it became rapidly obvious that a variety of viruses were circulating in the community and that an overestimation of myxovirus infection was at stake [8, 9, 10, 11] . As a better knowledge of the epidemic status was a key feature for national healthcare organization, hospital preparedness, patient management and disease control, unambiguous viral diagnosis appeared critical.", "As a better knowledge of the epidemic status was a key feature for national healthcare organization, hospital preparedness, patient management and disease control, unambiguous viral diagnosis appeared critical. In France, data on viral aetiologies associated with ILI were at best sporadic and correlations with clinical symptoms were often lacking. Extensive molecular assays to screening for respiratory viruses were not available countrywide for routine diagnosis.", "Extensive molecular assays to screening for respiratory viruses were not available countrywide for routine diagnosis. Therefore the epidemiological pattern of respiratory pathogens with overlapping seasonality was poorly known. The aim of the present study was to investigate respiratory pathogens involved in ILI during the early weeks of the 2009-2010 H1N1v diffusion in France (weeks 35 through 44) and describe the associated symptoms in paediatric and adult populations.", "The aim of the present study was to investigate respiratory pathogens involved in ILI during the early weeks of the 2009-2010 H1N1v diffusion in France (weeks 35 through 44) and describe the associated symptoms in paediatric and adult populations. This study was a non-interventional study with no addition to usual proceedures. Biological material and clinical data were obtained only for standard viral diagnostic following physicians' prescriptions (no specific sampling, no modification of the sampling protocol, no supplementary question in the national standardized questionnaire).", "Biological material and clinical data were obtained only for standard viral diagnostic following physicians' prescriptions (no specific sampling, no modification of the sampling protocol, no supplementary question in the national standardized questionnaire). Data analyses were carried out using an anonymized database. According to the French Health Public Law (CSP Art L 1121-1.1), such protocol does not require approval of an ethics committee and is exempted from informed consent application.", "According to the French Health Public Law (CSP Art L 1121-1.1), such protocol does not require approval of an ethics committee and is exempted from informed consent application. In the two academic hospitals, Saint-Louis hospital (SLS) in Paris and Tours hospital (TRS), influenza-like illness (ILI) was defined as a patient suffering from at least one general symptom (fever above 38uC, asthenia, myalgia, shivers or headache) and one respiratory symptom (cough, dyspnoea, rhinitis or pharyngitis), in agreement with the guidelines from the French Institut de Veille Sanitaire (InVS), a governmental institution responsible for surveillance and alert in all domains of public health [12] . Criteria for severe clinical presentation were temperature below 35uC or above 39uC despite antipyretic, cardiac frequency above 120/min, respiratory frequency above 30/min, respiratory distress, systolic arterial pressure below 90 mmHg or altered consciousness.", "Criteria for severe clinical presentation were temperature below 35uC or above 39uC despite antipyretic, cardiac frequency above 120/min, respiratory frequency above 30/min, respiratory distress, systolic arterial pressure below 90 mmHg or altered consciousness. Predisposing factors of critical illness were children younger than one year old, pregnant women, diabetes, chronic pre-existing disease (such as respiratory, cardiovascular, neurologic, renal, hepatic or hematologic diseases) and immunosuppression (associated with HIV infection, organ or hematopoietic stem cells transplantation, receipt of chemotherapy or corticosteroids) [13, 14] . A cluster of suspected influenza infections was defined as at least three possible cases in a week in a closed community (household, school,…) [15] .", "A cluster of suspected influenza infections was defined as at least three possible cases in a week in a closed community (household, school,…) [15] . In the two institutions, the prescription of H1N1v molecular testing was recommended for patients with ILI and with either a severe clinical presentation, an underlying risk factor of complications or a condition which was not improving under antiviral treatment. Investigation of grouped suspected cases was also recommended.", "Investigation of grouped suspected cases was also recommended. From week 35 (last week of August) to 44 (last week of October), 413 endonasal swabs were collected in 3 ml of Universal Transport Medium (Copan Diagnostics Inc, Murrieta, CA) from adults and children seen in emergency rooms for suspected ILI (Table 1 ) and sent to SLS and TRS laboratories for H1N1v detection. The two microbiology laboratories participated in the reference laboratories network for the detection of pandemic influenza H1N1v.", "The two microbiology laboratories participated in the reference laboratories network for the detection of pandemic influenza H1N1v. Clinical data were collected at the time of medical attention and reported by clinicians on a national standardized questionnaire provided by InVS [1, 12] . This questionnaire included the presence or absence of the main general and respiratory symptoms associated with ILI (fever, asthenia, myalgia, shivers, headache, cough, rhinitis, pharyngitis, sudden onset) [12] .", "This questionnaire included the presence or absence of the main general and respiratory symptoms associated with ILI (fever, asthenia, myalgia, shivers, headache, cough, rhinitis, pharyngitis, sudden onset) [12] . Total nucleic acid was extracted from 400 mL of Universal Transport Medium using the EasyMag System (Biomérieux, Marcy l'Etoile, France) in SLS or the EZ1 Advanced XL (Qiagen, Courtaboeuf, France) in TRS, according to the manufacturers' instructions (elution volume: 100 mL in SLS or 90 mL in TRS). Before extraction, 5 ml of an Internal Amplification Control (IAC) which contained an encephalomyocarditis virus (EMC) RNA transcript was added into the sample.", "Before extraction, 5 ml of an Internal Amplification Control (IAC) which contained an encephalomyocarditis virus (EMC) RNA transcript was added into the sample. Pandemic H1N1v infection was diagnosed by real-time reverse transcription-PCR (RT-PCR) assay on a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) according to the protocol of the Centers for Disease Control (CDC) [16] . Other respiratory infections were investigated by a multiplex molecular assay based on the Multiplex Ligation-dependent Probe-Amplification (MLPA) technology (RespiFinder19H, Pathofinder, Maastricht, The Netherlands) that allows the detection and differentiation of 14 respiratory viruses, including influenza virus A (InfA), influenza virus B (InfB), rhinovirus (RHV), parainfluenza viruses 1 to 4 (PIV-1 to PIV-4), human metapneumovirus (hMPV), adenovirus (ADV), respiratory syncytial virus A (RSVA), respiratory syncytial virus B (RSVB) and human coronaviruses 229E, OC43 and NL63 (Cor-229E, Cor-OC43, Cor-NL63) [17] .", "Other respiratory infections were investigated by a multiplex molecular assay based on the Multiplex Ligation-dependent Probe-Amplification (MLPA) technology (RespiFinder19H, Pathofinder, Maastricht, The Netherlands) that allows the detection and differentiation of 14 respiratory viruses, including influenza virus A (InfA), influenza virus B (InfB), rhinovirus (RHV), parainfluenza viruses 1 to 4 (PIV-1 to PIV-4), human metapneumovirus (hMPV), adenovirus (ADV), respiratory syncytial virus A (RSVA), respiratory syncytial virus B (RSVB) and human coronaviruses 229E, OC43 and NL63 (Cor-229E, Cor-OC43, Cor-NL63) [17] . The test allows also the detection of H5N1 influenza A virus and of four bacteria: Chlamydophila pneumoniae (CP), Mycoplasma pneumoniae (MP), Legionella pneumophila (LP) and Bordetella pertussis (BP). The amplified MLPA products were analyzed on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA).", "The amplified MLPA products were analyzed on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA). Fragment sizing analysis was performed with the GeneMarker software (SoftGenetics, LLC, State College, PA). Further testing for H1N1v was carried out with Simplexa TM Influenza A H1N1 (2009) (Focus Diagnostics, Cypress, California) when the CDC real time RT-PCR assay was negative for H1N1 and the RespiFinder19H assay was positive for Influenza A.", "Further testing for H1N1v was carried out with Simplexa TM Influenza A H1N1 (2009) (Focus Diagnostics, Cypress, California) when the CDC real time RT-PCR assay was negative for H1N1 and the RespiFinder19H assay was positive for Influenza A. If this latter assay was negative, H3N2 typing was performed as previously described [18] . Data from our study are summarized as frequencies and percentages for categorical variables.", "Data from our study are summarized as frequencies and percentages for categorical variables. Quantitative variables are presented as medians, 25th and 75th percentiles. To compare those variables according to the viral infection status, Fisher tests \n\nBy using CDC reference assay, H1N1v was detected in 66 samples out of 413 (16.6%), more frequently in SLS (38 samples) than in TRS (28 samples) (p,10 24 ).", "To compare those variables according to the viral infection status, Fisher tests \n\nBy using CDC reference assay, H1N1v was detected in 66 samples out of 413 (16.6%), more frequently in SLS (38 samples) than in TRS (28 samples) (p,10 24 ). Overall, weekly percentage of H1N1v positive endonasal swabs remained under 10% until week 41 and increase significantly after (P Trend ,0.0001) ( Figure 1 ). Rate of H1N1v detection reached 30% in SLS at week 42 and in TRS at week 44.", "Rate of H1N1v detection reached 30% in SLS at week 42 and in TRS at week 44. Overall, this rate was in agreement with results provided by the GROG network, showing an earlier start of H1N1v epidemic in Paris area [7, 19] . All 413 nucleic acid extracts were analyzed using the RespiFinder19H assay ( Figure 2 ).", "All 413 nucleic acid extracts were analyzed using the RespiFinder19H assay ( Figure 2 ). Sixty six patients tested H1N1v positive with CDC real time RT-PCR assay were confirmed with the multiplex assay. Thirteen were also co-infected by one or two other respiratory pathogens (multiple infections) ( Figure 2 ).", "Thirteen were also co-infected by one or two other respiratory pathogens (multiple infections) ( Figure 2 ). Three of the 347 H1N1v negative samples could not be studied with the multiplex assay because they contained RT-PCR inhibitors (no amplification of the internal control). Two hundred and fifteen (62.5%) of the remaining 344 H1N1v negative samples were found positive for at least one respiratory pathogen ( Figure 2 ).", "Two hundred and fifteen (62.5%) of the remaining 344 H1N1v negative samples were found positive for at least one respiratory pathogen ( Figure 2 ). Two hundred and twelve were positive for non influenza pathogens (189 single infections and 23 mixed infections with two, three or four viruses) and three additional single infections by influenza A were identified in SLS, including two by pandemic H1N1v and one by seasonal H3N2, as determined after molecular typing (data not shown). Overall, 68 patients (16.5%) were then positive for H1N1v, one for H3N2 and 212 for non influenza pathogens.", "Overall, 68 patients (16.5%) were then positive for H1N1v, one for H3N2 and 212 for non influenza pathogens. There were 245 single infections (55 with H1N1v and 190 with other respiratory pathogens) and 36 mixed infections (13 with H1N1v and 23 without H1N1v) ( Figure 2 ). Among H1N1v negative single infections, the most prevalent viruses were rhinovirus (62.6%, 119 patients), followed by parainfluenza viruses 1 to 4 (24.2%, 46 patients), adenovirus (5.3%, 10 patients), human coronavirus 229E, OC43 and NL63 (3.2%, 6 patients) and respiratory syncytial virus A and B (2.6%, 5 patients) (Figure 2 ).", "Among H1N1v negative single infections, the most prevalent viruses were rhinovirus (62.6%, 119 patients), followed by parainfluenza viruses 1 to 4 (24.2%, 46 patients), adenovirus (5.3%, 10 patients), human coronavirus 229E, OC43 and NL63 (3.2%, 6 patients) and respiratory syncytial virus A and B (2.6%, 5 patients) (Figure 2 ). In addition, RespiFinder19H assay identified three patients with bacterial infection, two with Mycoplasma pneumoniae (one 25 years old female in SLS and one 39 years old female in TRS) and one with Bordetella pertussis (one 60 years old male in SLS). No single infection by influenza B, hMPV, Chlamydophila pneumoniae or Legionella pneumophila was identified ( Figure 2 To analyze if viral co-infections occurred more frequently for some viruses, we carried out a two by two comparisons, that showed a higher proportion of co-infection only for ADV (p = 0.05).", "No single infection by influenza B, hMPV, Chlamydophila pneumoniae or Legionella pneumophila was identified ( Figure 2 To analyze if viral co-infections occurred more frequently for some viruses, we carried out a two by two comparisons, that showed a higher proportion of co-infection only for ADV (p = 0.05). Non-influenza respiratory viruses presented a different epidemic profile compared to H1N1v. Overall, in both hospitals, weekly rate of non-H1N1v respiratory viruses whether alone or involved in co-infection increased between week 37 and 39 (from 51.4% to 81.3%) and then consistently decreased ( Figure 3 ).", "Overall, in both hospitals, weekly rate of non-H1N1v respiratory viruses whether alone or involved in co-infection increased between week 37 and 39 (from 51.4% to 81.3%) and then consistently decreased ( Figure 3 ). RHV infections that represented nearly half of non-H1N1v viral infections (141 out of 213, 66.2%) were a significant contributing factor. In both hospitals, emergence of H1N1v cases was associated with a rapid decline of RHV rate of infection from 50-60% down to less than 20% with a one to two weeks gap between SLS and TRS.", "In both hospitals, emergence of H1N1v cases was associated with a rapid decline of RHV rate of infection from 50-60% down to less than 20% with a one to two weeks gap between SLS and TRS. Data on age ( In both institutions, 85.5% (106/124) children younger than 15 years of age were infected by at least one respiratory pathogen ( Table 2 ). H1N1v infected patients were not significantly younger than H1N1v non infected patients (27 years old vs. 25 years old, p = 0.80) (Figure 4) .", "H1N1v infected patients were not significantly younger than H1N1v non infected patients (27 years old vs. 25 years old, p = 0.80) (Figure 4) . However, 70.6% (48/68) of H1N1v cases were identified in patients under 40 years old (22 in SLS and 26 in TRS) and no case was observed in patients older than 65 years ( Table 2) . PIV infection occurred in very young patients (median (Figure 4) .", "PIV infection occurred in very young patients (median (Figure 4) . Consequently, PIV and ADV were more frequently detected in the younger population of TRS versus SLS (p,10 24 and p,10 23 respectively). In contrast, although individuals with RHV infection were slightly younger than individuals without (median age = 24 vs. 29 for patients without RHV, p = 0.05) (Figure 4) , influenza-like illness associated with RHV was more frequent in SLS than in TRS (p = 0.012).", "In contrast, although individuals with RHV infection were slightly younger than individuals without (median age = 24 vs. 29 for patients without RHV, p = 0.05) (Figure 4) , influenza-like illness associated with RHV was more frequent in SLS than in TRS (p = 0.012). Finally, patients with viral multiple infection were significantly younger than those with single infection (median, IDR: 4, 2-18.5 vs. 25, 6-43) and rates of mixed infection At the time of medical attention, 383 (92.7%) standardized clinical questionnaires were collected out of 413 patients. Four of them could not be exploited because they were too incomplete.", "Four of them could not be exploited because they were too incomplete. A review of the 379 workable questionnaires showed that 90.8% (344/379) of the patients included in this study fulfilled the criteria of ILI as defined above, and 52.5% had either a severe clinical presentation or an underlying risk factor of complications (45.9%, 174/379), or were in a suspected cluster of grouped cases (6.6%, 25/379). Overall, most patients have fever (93.9%) and cough (86.1%) ( Table 3) .", "Overall, most patients have fever (93.9%) and cough (86.1%) ( Table 3) . Other classical clinical signs associated with ILI such as asthenia, myalgia, shivers, headache, rhinitis or pharyngitis were less frequent. A sudden onset was also described in 59.2% of cases.", "A sudden onset was also described in 59.2% of cases. Only 32.5% of the patients had a temperature above 39uC; the age of these patients ranged from zero to 86 years, with a median age of 32 years and a mean age of 34 years (data not shown). In H1N1v infected patients (including single and multiple infections), the main symptoms were also fever (98.2%) and cough (89.5%) ( We then compared clinical characteristics between patients positive for H1N1v, patients positive for other respiratory pathogens and negative for H1N1v and patients without any detection of respiratory pathogens (as detected with RespiFin-der19H) ( Table 3 ).", "In H1N1v infected patients (including single and multiple infections), the main symptoms were also fever (98.2%) and cough (89.5%) ( We then compared clinical characteristics between patients positive for H1N1v, patients positive for other respiratory pathogens and negative for H1N1v and patients without any detection of respiratory pathogens (as detected with RespiFin-der19H) ( Table 3 ). There was no difference between the three groups except for fever, cough, pharyngitis. However for these latter symptoms, the comparison between patients positive for H1N1v and those positive for other respiratory pathogens or between patients positive for H1N1v and those without any detection of respiratory pathogens, showed no difference except for pharyngitis, which was less frequent in patients positive for H1N1v than in patients positive for other respiratory pathogens ( Table 3) .", "However for these latter symptoms, the comparison between patients positive for H1N1v and those positive for other respiratory pathogens or between patients positive for H1N1v and those without any detection of respiratory pathogens, showed no difference except for pharyngitis, which was less frequent in patients positive for H1N1v than in patients positive for other respiratory pathogens ( Table 3) . As RHV was the most frequent aetiology in ILI, we also compared clinical symptoms observed in patients with a single infection by RHV or by H1N1v (data not shown). There was no difference except that rhinitis and pharyngitis were significantly more frequent in RHV infection (62.7% vs. 34.1% [p = 0.006] and 39.0% vs. 10.0% [p = 0.001], respectively).", "There was no difference except that rhinitis and pharyngitis were significantly more frequent in RHV infection (62.7% vs. 34.1% [p = 0.006] and 39.0% vs. 10.0% [p = 0.001], respectively). Viral multiple infection (including samples with H1N1v) was not associated with a different clinical presentation. Fever and cough were observed in over 90% of the patients (90.6% and 90.3%, respectively), but only 33.3% of these patients had a temperature above 39uC, which was not different from patients with single viral infection (28.6%).", "Fever and cough were observed in over 90% of the patients (90.6% and 90.3%, respectively), but only 33.3% of these patients had a temperature above 39uC, which was not different from patients with single viral infection (28.6%). Our results highlight the high frequency of non-influenza viruses involved in acute respiratory infections during the epidemic period of a flu alert as defined by the Réseau Sentinelles according to ILI definition (a sudden fever above 39uC accompanied by myalgia and respiratory signs). These data extent previous observations in Europe reporting high prevalence of RHV infections before seasonal influenza [4, 20] or in 2009, before H1N1v pandemic influenza [1, 8, 9, 11, 21] .", "These data extent previous observations in Europe reporting high prevalence of RHV infections before seasonal influenza [4, 20] or in 2009, before H1N1v pandemic influenza [1, 8, 9, 11, 21] . We confirm that RHV represent the most frequent aetiology of acute respiratory Table 2 . Age of patients with respiratory samples positive for H1N1v, positive for other respiratory pathogens or negative.", "Age of patients with respiratory samples positive for H1N1v, positive for other respiratory pathogens or negative. infections both in adult and paediatric populations and may represent more than 50% of cases. We show that other viral infections than influenza and RHV may represent up to 30% of aetiologies.", "We show that other viral infections than influenza and RHV may represent up to 30% of aetiologies. We observed differences between the two hospitals, with a higher frequency of parainfluenza and ADV infections in Tours in contrast with a higher frequency of RHV in Paris, likely explained by the higher proportion of paediatric samples collected in Tours. However, despite the distance between the two institutions (about 250 km) and differences between the two populations, both presented similar patterns of high frequency of non-influenza viruses in acute respiratory infections before the flu epidemic wave and a decline when influenza reached epidemic levels.", "However, despite the distance between the two institutions (about 250 km) and differences between the two populations, both presented similar patterns of high frequency of non-influenza viruses in acute respiratory infections before the flu epidemic wave and a decline when influenza reached epidemic levels. In the two cities, high frequencies of RHV were seen at the same level with a likely different evolution speed, with sudden increase and decrease in SLS and more progressive variation in TRS. In both institutions, there was a decrease in the proportion and number of RHV diagnoses roughly in parallel with the increase of influenza diagnoses.", "In both institutions, there was a decrease in the proportion and number of RHV diagnoses roughly in parallel with the increase of influenza diagnoses. Indeed, H1N1v exceeds 20% of positive detection's rate only when RHV dropped under 40%. These data are thus consistent with negative interaction of the two epidemics at the population level.", "These data are thus consistent with negative interaction of the two epidemics at the population level. It was previously hypothesised that RHV epidemic could interfere with the spread of pandemic influenza [20, 21, 22] . Few in vitro data support this hypothesis.", "Few in vitro data support this hypothesis. It has been reported that interferon and other cytokines production by RHV infected cells induced a refractory state to virus infection These data include the three patients whose respiratory samples could not be studied with the multiplex assay because of RT-PCR inhibitors. of neighbouring cells [23] .", "of neighbouring cells [23] . Further work is needed to confirm in vitro and in vivo such negative interactions and if viral interference are really translated to a population level. Analysis of rhinovirus and influenza epidemics in previous years should also help to determine if similar interferences were observed with seasonal influenza and to elaborate modelling and prediction of the spread of influenza according to respiratory viruses' circulation.", "Analysis of rhinovirus and influenza epidemics in previous years should also help to determine if similar interferences were observed with seasonal influenza and to elaborate modelling and prediction of the spread of influenza according to respiratory viruses' circulation. Systematic extensive screening of respiratory viruses at a national level should be implemented for this purpose. Very few RSV infections were observed in contrast to usual epidemiology which was characterized the last four past years by a start of epidemics in weeks 44-45 [1] .", "Very few RSV infections were observed in contrast to usual epidemiology which was characterized the last four past years by a start of epidemics in weeks 44-45 [1] . It has been confirmed by other laboratories and the French InVS that the 2009-10 RSV epidemic was delayed and had a lower impact compared with the previous winter season [1, 24] . Delayed and reduced RSV spread may be due to viral interference between RSV and influenza.", "Delayed and reduced RSV spread may be due to viral interference between RSV and influenza. Another possible explanation is better prevention behaviour about respiratory infections as recommended by a national campaign including recommendations for hands washing after sneezing and the use of mask [1] . Influenza infections were mainly detected in patient under 40 years old and no case was found in patients older than 65.", "Influenza infections were mainly detected in patient under 40 years old and no case was found in patients older than 65. These results corroborate previous data suggesting that past seasonal H1N1 infections or vaccination may give partial crossed protection [10, 13, 25] . We have previously shown that the neutralizing titers against pandemic H1N1v virus correlate significantly with neutralizing titers against a seasonal H1N1 virus, and that the H1N1v pandemic influenza virus neutralizing titer was significantly higher in subjects who had recently been inoculated by a seasonal trivalent influenza vaccine [26] .", "We have previously shown that the neutralizing titers against pandemic H1N1v virus correlate significantly with neutralizing titers against a seasonal H1N1 virus, and that the H1N1v pandemic influenza virus neutralizing titer was significantly higher in subjects who had recently been inoculated by a seasonal trivalent influenza vaccine [26] . Viral co-infections were predominantly seen in paediatric patients, as previously described [4, 27, 28, 29] , both in influenza and non-influenza cases at a similar rate. No evidence of more pronounced respiratory impact was seen in these patients.", "No evidence of more pronounced respiratory impact was seen in these patients. Our results showed the lack of specific clinical signs associated with proven H1N1v infections. Clinical characteristics did not differ between influenza infections or other viral infections. In particular, the proportion of patients with fever above 39uC was not higher in H1N1v positive patients.", "In particular, the proportion of patients with fever above 39uC was not higher in H1N1v positive patients. In addition, the patients without any evidence of respiratory viral infections did not have different symptoms. These patients may have been infected with other virus not included in the multiplex assay (human Bocavirus, coronavirus HKU1) [9, 10, 11] or were seen too late at the time of viral shedding was cleared [30] .", "These patients may have been infected with other virus not included in the multiplex assay (human Bocavirus, coronavirus HKU1) [9, 10, 11] or were seen too late at the time of viral shedding was cleared [30] . However, to determine how specific the symptoms are for influenza would require to assess also the distribution of respiratory pathogens (H1N1v and other respiratory viruses) and related symptoms in patients presented at the emergency departments in SLS and TRS with respiratory syndromes, but not tested for H1N1v. In addition, despite some underlying conditions that were associated with complications not previously observed in seasonal influenza, most illnesses caused by the H1N1v virus were acute and self-limited [13, 31] .", "In addition, despite some underlying conditions that were associated with complications not previously observed in seasonal influenza, most illnesses caused by the H1N1v virus were acute and self-limited [13, 31] . The higher proportion of non influenza viruses reported in ILI in 2009 was thus most likely a consequence of more frequent visits to a doctor for respiratory tract infections than usually observed for fear of the flu pandemic. The general lack of difference in symptoms in the particular context of H1N1v pandemic has therefore to be considered with caution and does not rule out that more significant differences may arise in future influenza epidemics with other influenza viruses.", "The general lack of difference in symptoms in the particular context of H1N1v pandemic has therefore to be considered with caution and does not rule out that more significant differences may arise in future influenza epidemics with other influenza viruses. Our data confirm that it may be virtually impossible to recognize symptoms heralding H1N1v infections and virological data should be helpful along with clinical reports to monitor influenza epidemic [10] . Molecular multiplex detection has recently emerged as a potent diagnostic tool to determine acute respiratory infections' aetiologies [11, 32, 33] .", "Molecular multiplex detection has recently emerged as a potent diagnostic tool to determine acute respiratory infections' aetiologies [11, 32, 33] . These data show that sensitive molecular multiplex detection of respiratory viruses is feasible and efficient for the detection of virus involved in acute respiratory infections and provides insights into their epidemic profile. Our results confirm the performance of RespiFinder19H assay to detecting respiratory viruses in the general population as recently shown in transplant patients with ILI [34] .", "Our results confirm the performance of RespiFinder19H assay to detecting respiratory viruses in the general population as recently shown in transplant patients with ILI [34] . RespiFinder19H confirmed all H1N1 infections detected by the CDC reference assay and was able to identify two additional H1N1 cases suggesting a high sensitivity of this multiplex assay to detect influenza A infections. In conclusion, our results highlight that successive and mixed outbreaks of respiratory viral infections may affect influenza epidemiology and can lead to misinterpret the early development of a flu epidemic.", "In conclusion, our results highlight that successive and mixed outbreaks of respiratory viral infections may affect influenza epidemiology and can lead to misinterpret the early development of a flu epidemic. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management." ]

[ "BACKGROUND: Influenza-like illness (ILI) may be caused by a variety of pathogens. Clinical observations are of little help to recognise myxovirus infection and implement appropriate prevention measures. The limited use of molecular tools underestimates the role of other common pathogens.", "The limited use of molecular tools underestimates the role of other common pathogens. OBJECTIVES: During the early weeks of the 2009–2010 flu pandemic, a clinical and virological survey was conducted in adult and paediatric patients with ILI referred to two French University hospitals in Paris and Tours. Aims were to investigate the different pathogens involved in ILI and describe the associated symptoms.", "Aims were to investigate the different pathogens involved in ILI and describe the associated symptoms. METHODS: H1N1v pandemic influenza diagnosis was performed with real time RT-PCR assay. Other viral aetiologies were investigated by the molecular multiplex assay RespiFinder19®. Clinical data were collected prospectively by physicians using a standard questionnaire.", "Clinical data were collected prospectively by physicians using a standard questionnaire. RESULTS: From week 35 to 44, endonasal swabs were collected in 413 patients. Overall, 68 samples (16.5%) were positive for H1N1v. In 13 of them, other respiratory pathogens were also detected.", "In 13 of them, other respiratory pathogens were also detected. Among H1N1v negative samples, 213 (61.9%) were positive for various respiratory agents, 190 in single infections and 23 in mixed infections. The most prevalent viruses in H1N1v negative single infections were rhinovirus (62.6%), followed by parainfluenza viruses (24.2%) and adenovirus (5.3%).", "The most prevalent viruses in H1N1v negative single infections were rhinovirus (62.6%), followed by parainfluenza viruses (24.2%) and adenovirus (5.3%). 70.6% of H1N1v cases were identified in patients under 40 years and none after 65 years. There was no difference between clinical symptoms observed in patients infected with H1N1v or with other pathogens.", "There was no difference between clinical symptoms observed in patients infected with H1N1v or with other pathogens. CONCLUSION: Our results highlight the high frequency of non-influenza viruses involved in ILI during the pre-epidemic period of a flu alert and the lack of specific clinical signs associated with influenza infections. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management.", "Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management. Text: In order to monitor the spread of influenza and alert health handlers, several epidemiological tools have been developed. In France, a network of 1300 general practitioners, ''Réseau Sentinelles'', working throughout the country, provides real-time clinical data used to evaluate regional and national influenza spreading [1, 2] .", "In France, a network of 1300 general practitioners, ''Réseau Sentinelles'', working throughout the country, provides real-time clinical data used to evaluate regional and national influenza spreading [1, 2] . The criteria used by this network to define clinical influenza-like illness (ILI) are the occurrence of a sudden fever above 39uC with myalgia and respiratory signs. In general no formal viral diagnosis is carried out.", "In general no formal viral diagnosis is carried out. The Groupes Régionaux d'Observation de la Grippe (GROG) is a second French network that surveys the emergence and the spread of the influenza viruses [3, 4] . This network is based on clinical surveillance of acute respiratory infections and laboratory analysis of nasal specimens collected from adults and children by volunteer general practitioners and pediatricians.", "This network is based on clinical surveillance of acute respiratory infections and laboratory analysis of nasal specimens collected from adults and children by volunteer general practitioners and pediatricians. According to the sentinel network's criteria, French health authorities proclaimed that flu epidemic level was reached during the second week of September 2009 (week 37) [5, 6] . On the contrary, data provided by the GROG showed only sporadic H1N1v activity until the last week of October (week 44) [6, 7] .", "On the contrary, data provided by the GROG showed only sporadic H1N1v activity until the last week of October (week 44) [6, 7] . Thus, it became rapidly obvious that a variety of viruses were circulating in the community and that an overestimation of myxovirus infection was at stake [8, 9, 10, 11] . As a better knowledge of the epidemic status was a key feature for national healthcare organization, hospital preparedness, patient management and disease control, unambiguous viral diagnosis appeared critical.", "As a better knowledge of the epidemic status was a key feature for national healthcare organization, hospital preparedness, patient management and disease control, unambiguous viral diagnosis appeared critical. In France, data on viral aetiologies associated with ILI were at best sporadic and correlations with clinical symptoms were often lacking. Extensive molecular assays to screening for respiratory viruses were not available countrywide for routine diagnosis.", "Extensive molecular assays to screening for respiratory viruses were not available countrywide for routine diagnosis. Therefore the epidemiological pattern of respiratory pathogens with overlapping seasonality was poorly known. The aim of the present study was to investigate respiratory pathogens involved in ILI during the early weeks of the 2009-2010 H1N1v diffusion in France (weeks 35 through 44) and describe the associated symptoms in paediatric and adult populations.", "The aim of the present study was to investigate respiratory pathogens involved in ILI during the early weeks of the 2009-2010 H1N1v diffusion in France (weeks 35 through 44) and describe the associated symptoms in paediatric and adult populations. This study was a non-interventional study with no addition to usual proceedures. Biological material and clinical data were obtained only for standard viral diagnostic following physicians' prescriptions (no specific sampling, no modification of the sampling protocol, no supplementary question in the national standardized questionnaire).", "Biological material and clinical data were obtained only for standard viral diagnostic following physicians' prescriptions (no specific sampling, no modification of the sampling protocol, no supplementary question in the national standardized questionnaire). Data analyses were carried out using an anonymized database. According to the French Health Public Law (CSP Art L 1121-1.1), such protocol does not require approval of an ethics committee and is exempted from informed consent application.", "According to the French Health Public Law (CSP Art L 1121-1.1), such protocol does not require approval of an ethics committee and is exempted from informed consent application. In the two academic hospitals, Saint-Louis hospital (SLS) in Paris and Tours hospital (TRS), influenza-like illness (ILI) was defined as a patient suffering from at least one general symptom (fever above 38uC, asthenia, myalgia, shivers or headache) and one respiratory symptom (cough, dyspnoea, rhinitis or pharyngitis), in agreement with the guidelines from the French Institut de Veille Sanitaire (InVS), a governmental institution responsible for surveillance and alert in all domains of public health [12] . Criteria for severe clinical presentation were temperature below 35uC or above 39uC despite antipyretic, cardiac frequency above 120/min, respiratory frequency above 30/min, respiratory distress, systolic arterial pressure below 90 mmHg or altered consciousness.", "Criteria for severe clinical presentation were temperature below 35uC or above 39uC despite antipyretic, cardiac frequency above 120/min, respiratory frequency above 30/min, respiratory distress, systolic arterial pressure below 90 mmHg or altered consciousness. Predisposing factors of critical illness were children younger than one year old, pregnant women, diabetes, chronic pre-existing disease (such as respiratory, cardiovascular, neurologic, renal, hepatic or hematologic diseases) and immunosuppression (associated with HIV infection, organ or hematopoietic stem cells transplantation, receipt of chemotherapy or corticosteroids) [13, 14] . A cluster of suspected influenza infections was defined as at least three possible cases in a week in a closed community (household, school,…) [15] .", "A cluster of suspected influenza infections was defined as at least three possible cases in a week in a closed community (household, school,…) [15] . In the two institutions, the prescription of H1N1v molecular testing was recommended for patients with ILI and with either a severe clinical presentation, an underlying risk factor of complications or a condition which was not improving under antiviral treatment. Investigation of grouped suspected cases was also recommended.", "Investigation of grouped suspected cases was also recommended. From week 35 (last week of August) to 44 (last week of October), 413 endonasal swabs were collected in 3 ml of Universal Transport Medium (Copan Diagnostics Inc, Murrieta, CA) from adults and children seen in emergency rooms for suspected ILI (Table 1 ) and sent to SLS and TRS laboratories for H1N1v detection. The two microbiology laboratories participated in the reference laboratories network for the detection of pandemic influenza H1N1v.", "The two microbiology laboratories participated in the reference laboratories network for the detection of pandemic influenza H1N1v. Clinical data were collected at the time of medical attention and reported by clinicians on a national standardized questionnaire provided by InVS [1, 12] . This questionnaire included the presence or absence of the main general and respiratory symptoms associated with ILI (fever, asthenia, myalgia, shivers, headache, cough, rhinitis, pharyngitis, sudden onset) [12] .", "This questionnaire included the presence or absence of the main general and respiratory symptoms associated with ILI (fever, asthenia, myalgia, shivers, headache, cough, rhinitis, pharyngitis, sudden onset) [12] . Total nucleic acid was extracted from 400 mL of Universal Transport Medium using the EasyMag System (Biomérieux, Marcy l'Etoile, France) in SLS or the EZ1 Advanced XL (Qiagen, Courtaboeuf, France) in TRS, according to the manufacturers' instructions (elution volume: 100 mL in SLS or 90 mL in TRS). Before extraction, 5 ml of an Internal Amplification Control (IAC) which contained an encephalomyocarditis virus (EMC) RNA transcript was added into the sample.", "Before extraction, 5 ml of an Internal Amplification Control (IAC) which contained an encephalomyocarditis virus (EMC) RNA transcript was added into the sample. Pandemic H1N1v infection was diagnosed by real-time reverse transcription-PCR (RT-PCR) assay on a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) according to the protocol of the Centers for Disease Control (CDC) [16] . Other respiratory infections were investigated by a multiplex molecular assay based on the Multiplex Ligation-dependent Probe-Amplification (MLPA) technology (RespiFinder19H, Pathofinder, Maastricht, The Netherlands) that allows the detection and differentiation of 14 respiratory viruses, including influenza virus A (InfA), influenza virus B (InfB), rhinovirus (RHV), parainfluenza viruses 1 to 4 (PIV-1 to PIV-4), human metapneumovirus (hMPV), adenovirus (ADV), respiratory syncytial virus A (RSVA), respiratory syncytial virus B (RSVB) and human coronaviruses 229E, OC43 and NL63 (Cor-229E, Cor-OC43, Cor-NL63) [17] .", "Other respiratory infections were investigated by a multiplex molecular assay based on the Multiplex Ligation-dependent Probe-Amplification (MLPA) technology (RespiFinder19H, Pathofinder, Maastricht, The Netherlands) that allows the detection and differentiation of 14 respiratory viruses, including influenza virus A (InfA), influenza virus B (InfB), rhinovirus (RHV), parainfluenza viruses 1 to 4 (PIV-1 to PIV-4), human metapneumovirus (hMPV), adenovirus (ADV), respiratory syncytial virus A (RSVA), respiratory syncytial virus B (RSVB) and human coronaviruses 229E, OC43 and NL63 (Cor-229E, Cor-OC43, Cor-NL63) [17] . The test allows also the detection of H5N1 influenza A virus and of four bacteria: Chlamydophila pneumoniae (CP), Mycoplasma pneumoniae (MP), Legionella pneumophila (LP) and Bordetella pertussis (BP). The amplified MLPA products were analyzed on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA).", "The amplified MLPA products were analyzed on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA). Fragment sizing analysis was performed with the GeneMarker software (SoftGenetics, LLC, State College, PA). Further testing for H1N1v was carried out with Simplexa TM Influenza A H1N1 (2009) (Focus Diagnostics, Cypress, California) when the CDC real time RT-PCR assay was negative for H1N1 and the RespiFinder19H assay was positive for Influenza A.", "Further testing for H1N1v was carried out with Simplexa TM Influenza A H1N1 (2009) (Focus Diagnostics, Cypress, California) when the CDC real time RT-PCR assay was negative for H1N1 and the RespiFinder19H assay was positive for Influenza A. If this latter assay was negative, H3N2 typing was performed as previously described [18] . Data from our study are summarized as frequencies and percentages for categorical variables.", "Data from our study are summarized as frequencies and percentages for categorical variables. Quantitative variables are presented as medians, 25th and 75th percentiles. To compare those variables according to the viral infection status, Fisher tests \n\nBy using CDC reference assay, H1N1v was detected in 66 samples out of 413 (16.6%), more frequently in SLS (38 samples) than in TRS (28 samples) (p,10 24 ).", "To compare those variables according to the viral infection status, Fisher tests \n\nBy using CDC reference assay, H1N1v was detected in 66 samples out of 413 (16.6%), more frequently in SLS (38 samples) than in TRS (28 samples) (p,10 24 ). Overall, weekly percentage of H1N1v positive endonasal swabs remained under 10% until week 41 and increase significantly after (P Trend ,0.0001) ( Figure 1 ). Rate of H1N1v detection reached 30% in SLS at week 42 and in TRS at week 44.", "Rate of H1N1v detection reached 30% in SLS at week 42 and in TRS at week 44. Overall, this rate was in agreement with results provided by the GROG network, showing an earlier start of H1N1v epidemic in Paris area [7, 19] . All 413 nucleic acid extracts were analyzed using the RespiFinder19H assay ( Figure 2 ).", "All 413 nucleic acid extracts were analyzed using the RespiFinder19H assay ( Figure 2 ). Sixty six patients tested H1N1v positive with CDC real time RT-PCR assay were confirmed with the multiplex assay. Thirteen were also co-infected by one or two other respiratory pathogens (multiple infections) ( Figure 2 ).", "Thirteen were also co-infected by one or two other respiratory pathogens (multiple infections) ( Figure 2 ). Three of the 347 H1N1v negative samples could not be studied with the multiplex assay because they contained RT-PCR inhibitors (no amplification of the internal control). Two hundred and fifteen (62.5%) of the remaining 344 H1N1v negative samples were found positive for at least one respiratory pathogen ( Figure 2 ).", "Two hundred and fifteen (62.5%) of the remaining 344 H1N1v negative samples were found positive for at least one respiratory pathogen ( Figure 2 ). Two hundred and twelve were positive for non influenza pathogens (189 single infections and 23 mixed infections with two, three or four viruses) and three additional single infections by influenza A were identified in SLS, including two by pandemic H1N1v and one by seasonal H3N2, as determined after molecular typing (data not shown). Overall, 68 patients (16.5%) were then positive for H1N1v, one for H3N2 and 212 for non influenza pathogens.", "Overall, 68 patients (16.5%) were then positive for H1N1v, one for H3N2 and 212 for non influenza pathogens. There were 245 single infections (55 with H1N1v and 190 with other respiratory pathogens) and 36 mixed infections (13 with H1N1v and 23 without H1N1v) ( Figure 2 ). Among H1N1v negative single infections, the most prevalent viruses were rhinovirus (62.6%, 119 patients), followed by parainfluenza viruses 1 to 4 (24.2%, 46 patients), adenovirus (5.3%, 10 patients), human coronavirus 229E, OC43 and NL63 (3.2%, 6 patients) and respiratory syncytial virus A and B (2.6%, 5 patients) (Figure 2 ).", "Among H1N1v negative single infections, the most prevalent viruses were rhinovirus (62.6%, 119 patients), followed by parainfluenza viruses 1 to 4 (24.2%, 46 patients), adenovirus (5.3%, 10 patients), human coronavirus 229E, OC43 and NL63 (3.2%, 6 patients) and respiratory syncytial virus A and B (2.6%, 5 patients) (Figure 2 ). In addition, RespiFinder19H assay identified three patients with bacterial infection, two with Mycoplasma pneumoniae (one 25 years old female in SLS and one 39 years old female in TRS) and one with Bordetella pertussis (one 60 years old male in SLS). No single infection by influenza B, hMPV, Chlamydophila pneumoniae or Legionella pneumophila was identified ( Figure 2 To analyze if viral co-infections occurred more frequently for some viruses, we carried out a two by two comparisons, that showed a higher proportion of co-infection only for ADV (p = 0.05).", "No single infection by influenza B, hMPV, Chlamydophila pneumoniae or Legionella pneumophila was identified ( Figure 2 To analyze if viral co-infections occurred more frequently for some viruses, we carried out a two by two comparisons, that showed a higher proportion of co-infection only for ADV (p = 0.05). Non-influenza respiratory viruses presented a different epidemic profile compared to H1N1v. Overall, in both hospitals, weekly rate of non-H1N1v respiratory viruses whether alone or involved in co-infection increased between week 37 and 39 (from 51.4% to 81.3%) and then consistently decreased ( Figure 3 ).", "Overall, in both hospitals, weekly rate of non-H1N1v respiratory viruses whether alone or involved in co-infection increased between week 37 and 39 (from 51.4% to 81.3%) and then consistently decreased ( Figure 3 ). RHV infections that represented nearly half of non-H1N1v viral infections (141 out of 213, 66.2%) were a significant contributing factor. In both hospitals, emergence of H1N1v cases was associated with a rapid decline of RHV rate of infection from 50-60% down to less than 20% with a one to two weeks gap between SLS and TRS.", "In both hospitals, emergence of H1N1v cases was associated with a rapid decline of RHV rate of infection from 50-60% down to less than 20% with a one to two weeks gap between SLS and TRS. Data on age ( In both institutions, 85.5% (106/124) children younger than 15 years of age were infected by at least one respiratory pathogen ( Table 2 ). H1N1v infected patients were not significantly younger than H1N1v non infected patients (27 years old vs. 25 years old, p = 0.80) (Figure 4) .", "H1N1v infected patients were not significantly younger than H1N1v non infected patients (27 years old vs. 25 years old, p = 0.80) (Figure 4) . However, 70.6% (48/68) of H1N1v cases were identified in patients under 40 years old (22 in SLS and 26 in TRS) and no case was observed in patients older than 65 years ( Table 2) . PIV infection occurred in very young patients (median (Figure 4) .", "PIV infection occurred in very young patients (median (Figure 4) . Consequently, PIV and ADV were more frequently detected in the younger population of TRS versus SLS (p,10 24 and p,10 23 respectively). In contrast, although individuals with RHV infection were slightly younger than individuals without (median age = 24 vs. 29 for patients without RHV, p = 0.05) (Figure 4) , influenza-like illness associated with RHV was more frequent in SLS than in TRS (p = 0.012).", "In contrast, although individuals with RHV infection were slightly younger than individuals without (median age = 24 vs. 29 for patients without RHV, p = 0.05) (Figure 4) , influenza-like illness associated with RHV was more frequent in SLS than in TRS (p = 0.012). Finally, patients with viral multiple infection were significantly younger than those with single infection (median, IDR: 4, 2-18.5 vs. 25, 6-43) and rates of mixed infection At the time of medical attention, 383 (92.7%) standardized clinical questionnaires were collected out of 413 patients. Four of them could not be exploited because they were too incomplete.", "Four of them could not be exploited because they were too incomplete. A review of the 379 workable questionnaires showed that 90.8% (344/379) of the patients included in this study fulfilled the criteria of ILI as defined above, and 52.5% had either a severe clinical presentation or an underlying risk factor of complications (45.9%, 174/379), or were in a suspected cluster of grouped cases (6.6%, 25/379). Overall, most patients have fever (93.9%) and cough (86.1%) ( Table 3) .", "Overall, most patients have fever (93.9%) and cough (86.1%) ( Table 3) . Other classical clinical signs associated with ILI such as asthenia, myalgia, shivers, headache, rhinitis or pharyngitis were less frequent. A sudden onset was also described in 59.2% of cases.", "A sudden onset was also described in 59.2% of cases. Only 32.5% of the patients had a temperature above 39uC; the age of these patients ranged from zero to 86 years, with a median age of 32 years and a mean age of 34 years (data not shown). In H1N1v infected patients (including single and multiple infections), the main symptoms were also fever (98.2%) and cough (89.5%) ( We then compared clinical characteristics between patients positive for H1N1v, patients positive for other respiratory pathogens and negative for H1N1v and patients without any detection of respiratory pathogens (as detected with RespiFin-der19H) ( Table 3 ).", "In H1N1v infected patients (including single and multiple infections), the main symptoms were also fever (98.2%) and cough (89.5%) ( We then compared clinical characteristics between patients positive for H1N1v, patients positive for other respiratory pathogens and negative for H1N1v and patients without any detection of respiratory pathogens (as detected with RespiFin-der19H) ( Table 3 ). There was no difference between the three groups except for fever, cough, pharyngitis. However for these latter symptoms, the comparison between patients positive for H1N1v and those positive for other respiratory pathogens or between patients positive for H1N1v and those without any detection of respiratory pathogens, showed no difference except for pharyngitis, which was less frequent in patients positive for H1N1v than in patients positive for other respiratory pathogens ( Table 3) .", "However for these latter symptoms, the comparison between patients positive for H1N1v and those positive for other respiratory pathogens or between patients positive for H1N1v and those without any detection of respiratory pathogens, showed no difference except for pharyngitis, which was less frequent in patients positive for H1N1v than in patients positive for other respiratory pathogens ( Table 3) . As RHV was the most frequent aetiology in ILI, we also compared clinical symptoms observed in patients with a single infection by RHV or by H1N1v (data not shown). There was no difference except that rhinitis and pharyngitis were significantly more frequent in RHV infection (62.7% vs. 34.1% [p = 0.006] and 39.0% vs. 10.0% [p = 0.001], respectively).", "There was no difference except that rhinitis and pharyngitis were significantly more frequent in RHV infection (62.7% vs. 34.1% [p = 0.006] and 39.0% vs. 10.0% [p = 0.001], respectively). Viral multiple infection (including samples with H1N1v) was not associated with a different clinical presentation. Fever and cough were observed in over 90% of the patients (90.6% and 90.3%, respectively), but only 33.3% of these patients had a temperature above 39uC, which was not different from patients with single viral infection (28.6%).", "Fever and cough were observed in over 90% of the patients (90.6% and 90.3%, respectively), but only 33.3% of these patients had a temperature above 39uC, which was not different from patients with single viral infection (28.6%). Our results highlight the high frequency of non-influenza viruses involved in acute respiratory infections during the epidemic period of a flu alert as defined by the Réseau Sentinelles according to ILI definition (a sudden fever above 39uC accompanied by myalgia and respiratory signs). These data extent previous observations in Europe reporting high prevalence of RHV infections before seasonal influenza [4, 20] or in 2009, before H1N1v pandemic influenza [1, 8, 9, 11, 21] .", "These data extent previous observations in Europe reporting high prevalence of RHV infections before seasonal influenza [4, 20] or in 2009, before H1N1v pandemic influenza [1, 8, 9, 11, 21] . We confirm that RHV represent the most frequent aetiology of acute respiratory Table 2 . Age of patients with respiratory samples positive for H1N1v, positive for other respiratory pathogens or negative.", "Age of patients with respiratory samples positive for H1N1v, positive for other respiratory pathogens or negative. infections both in adult and paediatric populations and may represent more than 50% of cases. We show that other viral infections than influenza and RHV may represent up to 30% of aetiologies.", "We show that other viral infections than influenza and RHV may represent up to 30% of aetiologies. We observed differences between the two hospitals, with a higher frequency of parainfluenza and ADV infections in Tours in contrast with a higher frequency of RHV in Paris, likely explained by the higher proportion of paediatric samples collected in Tours. However, despite the distance between the two institutions (about 250 km) and differences between the two populations, both presented similar patterns of high frequency of non-influenza viruses in acute respiratory infections before the flu epidemic wave and a decline when influenza reached epidemic levels.", "However, despite the distance between the two institutions (about 250 km) and differences between the two populations, both presented similar patterns of high frequency of non-influenza viruses in acute respiratory infections before the flu epidemic wave and a decline when influenza reached epidemic levels. In the two cities, high frequencies of RHV were seen at the same level with a likely different evolution speed, with sudden increase and decrease in SLS and more progressive variation in TRS. In both institutions, there was a decrease in the proportion and number of RHV diagnoses roughly in parallel with the increase of influenza diagnoses.", "In both institutions, there was a decrease in the proportion and number of RHV diagnoses roughly in parallel with the increase of influenza diagnoses. Indeed, H1N1v exceeds 20% of positive detection's rate only when RHV dropped under 40%. These data are thus consistent with negative interaction of the two epidemics at the population level.", "These data are thus consistent with negative interaction of the two epidemics at the population level. It was previously hypothesised that RHV epidemic could interfere with the spread of pandemic influenza [20, 21, 22] . Few in vitro data support this hypothesis.", "Few in vitro data support this hypothesis. It has been reported that interferon and other cytokines production by RHV infected cells induced a refractory state to virus infection These data include the three patients whose respiratory samples could not be studied with the multiplex assay because of RT-PCR inhibitors. of neighbouring cells [23] .", "of neighbouring cells [23] . Further work is needed to confirm in vitro and in vivo such negative interactions and if viral interference are really translated to a population level. Analysis of rhinovirus and influenza epidemics in previous years should also help to determine if similar interferences were observed with seasonal influenza and to elaborate modelling and prediction of the spread of influenza according to respiratory viruses' circulation.", "Analysis of rhinovirus and influenza epidemics in previous years should also help to determine if similar interferences were observed with seasonal influenza and to elaborate modelling and prediction of the spread of influenza according to respiratory viruses' circulation. Systematic extensive screening of respiratory viruses at a national level should be implemented for this purpose. Very few RSV infections were observed in contrast to usual epidemiology which was characterized the last four past years by a start of epidemics in weeks 44-45 [1] .", "Very few RSV infections were observed in contrast to usual epidemiology which was characterized the last four past years by a start of epidemics in weeks 44-45 [1] . It has been confirmed by other laboratories and the French InVS that the 2009-10 RSV epidemic was delayed and had a lower impact compared with the previous winter season [1, 24] . Delayed and reduced RSV spread may be due to viral interference between RSV and influenza.", "Delayed and reduced RSV spread may be due to viral interference between RSV and influenza. Another possible explanation is better prevention behaviour about respiratory infections as recommended by a national campaign including recommendations for hands washing after sneezing and the use of mask [1] . Influenza infections were mainly detected in patient under 40 years old and no case was found in patients older than 65.", "Influenza infections were mainly detected in patient under 40 years old and no case was found in patients older than 65. These results corroborate previous data suggesting that past seasonal H1N1 infections or vaccination may give partial crossed protection [10, 13, 25] . We have previously shown that the neutralizing titers against pandemic H1N1v virus correlate significantly with neutralizing titers against a seasonal H1N1 virus, and that the H1N1v pandemic influenza virus neutralizing titer was significantly higher in subjects who had recently been inoculated by a seasonal trivalent influenza vaccine [26] .", "We have previously shown that the neutralizing titers against pandemic H1N1v virus correlate significantly with neutralizing titers against a seasonal H1N1 virus, and that the H1N1v pandemic influenza virus neutralizing titer was significantly higher in subjects who had recently been inoculated by a seasonal trivalent influenza vaccine [26] . Viral co-infections were predominantly seen in paediatric patients, as previously described [4, 27, 28, 29] , both in influenza and non-influenza cases at a similar rate. No evidence of more pronounced respiratory impact was seen in these patients.", "No evidence of more pronounced respiratory impact was seen in these patients. Our results showed the lack of specific clinical signs associated with proven H1N1v infections. Clinical characteristics did not differ between influenza infections or other viral infections. In particular, the proportion of patients with fever above 39uC was not higher in H1N1v positive patients.", "In particular, the proportion of patients with fever above 39uC was not higher in H1N1v positive patients. In addition, the patients without any evidence of respiratory viral infections did not have different symptoms. These patients may have been infected with other virus not included in the multiplex assay (human Bocavirus, coronavirus HKU1) [9, 10, 11] or were seen too late at the time of viral shedding was cleared [30] .", "These patients may have been infected with other virus not included in the multiplex assay (human Bocavirus, coronavirus HKU1) [9, 10, 11] or were seen too late at the time of viral shedding was cleared [30] . However, to determine how specific the symptoms are for influenza would require to assess also the distribution of respiratory pathogens (H1N1v and other respiratory viruses) and related symptoms in patients presented at the emergency departments in SLS and TRS with respiratory syndromes, but not tested for H1N1v. In addition, despite some underlying conditions that were associated with complications not previously observed in seasonal influenza, most illnesses caused by the H1N1v virus were acute and self-limited [13, 31] .", "In addition, despite some underlying conditions that were associated with complications not previously observed in seasonal influenza, most illnesses caused by the H1N1v virus were acute and self-limited [13, 31] . The higher proportion of non influenza viruses reported in ILI in 2009 was thus most likely a consequence of more frequent visits to a doctor for respiratory tract infections than usually observed for fear of the flu pandemic. The general lack of difference in symptoms in the particular context of H1N1v pandemic has therefore to be considered with caution and does not rule out that more significant differences may arise in future influenza epidemics with other influenza viruses.", "The general lack of difference in symptoms in the particular context of H1N1v pandemic has therefore to be considered with caution and does not rule out that more significant differences may arise in future influenza epidemics with other influenza viruses. Our data confirm that it may be virtually impossible to recognize symptoms heralding H1N1v infections and virological data should be helpful along with clinical reports to monitor influenza epidemic [10] . Molecular multiplex detection has recently emerged as a potent diagnostic tool to determine acute respiratory infections' aetiologies [11, 32, 33] .", "Molecular multiplex detection has recently emerged as a potent diagnostic tool to determine acute respiratory infections' aetiologies [11, 32, 33] . These data show that sensitive molecular multiplex detection of respiratory viruses is feasible and efficient for the detection of virus involved in acute respiratory infections and provides insights into their epidemic profile. Our results confirm the performance of RespiFinder19H assay to detecting respiratory viruses in the general population as recently shown in transplant patients with ILI [34] .", "Our results confirm the performance of RespiFinder19H assay to detecting respiratory viruses in the general population as recently shown in transplant patients with ILI [34] . RespiFinder19H confirmed all H1N1 infections detected by the CDC reference assay and was able to identify two additional H1N1 cases suggesting a high sensitivity of this multiplex assay to detect influenza A infections. In conclusion, our results highlight that successive and mixed outbreaks of respiratory viral infections may affect influenza epidemiology and can lead to misinterpret the early development of a flu epidemic.", "In conclusion, our results highlight that successive and mixed outbreaks of respiratory viral infections may affect influenza epidemiology and can lead to misinterpret the early development of a flu epidemic. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management." ]

[ "BACKGROUND: Influenza-like illness (ILI) may be caused by a variety of pathogens. Clinical observations are of little help to recognise myxovirus infection and implement appropriate prevention measures. The limited use of molecular tools underestimates the role of other common pathogens.", "The limited use of molecular tools underestimates the role of other common pathogens. OBJECTIVES: During the early weeks of the 2009–2010 flu pandemic, a clinical and virological survey was conducted in adult and paediatric patients with ILI referred to two French University hospitals in Paris and Tours. Aims were to investigate the different pathogens involved in ILI and describe the associated symptoms.", "Aims were to investigate the different pathogens involved in ILI and describe the associated symptoms. METHODS: H1N1v pandemic influenza diagnosis was performed with real time RT-PCR assay. Other viral aetiologies were investigated by the molecular multiplex assay RespiFinder19®. Clinical data were collected prospectively by physicians using a standard questionnaire.", "Clinical data were collected prospectively by physicians using a standard questionnaire. RESULTS: From week 35 to 44, endonasal swabs were collected in 413 patients. Overall, 68 samples (16.5%) were positive for H1N1v. In 13 of them, other respiratory pathogens were also detected.", "In 13 of them, other respiratory pathogens were also detected. Among H1N1v negative samples, 213 (61.9%) were positive for various respiratory agents, 190 in single infections and 23 in mixed infections. The most prevalent viruses in H1N1v negative single infections were rhinovirus (62.6%), followed by parainfluenza viruses (24.2%) and adenovirus (5.3%).", "The most prevalent viruses in H1N1v negative single infections were rhinovirus (62.6%), followed by parainfluenza viruses (24.2%) and adenovirus (5.3%). 70.6% of H1N1v cases were identified in patients under 40 years and none after 65 years. There was no difference between clinical symptoms observed in patients infected with H1N1v or with other pathogens.", "There was no difference between clinical symptoms observed in patients infected with H1N1v or with other pathogens. CONCLUSION: Our results highlight the high frequency of non-influenza viruses involved in ILI during the pre-epidemic period of a flu alert and the lack of specific clinical signs associated with influenza infections. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management.", "Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management. Text: In order to monitor the spread of influenza and alert health handlers, several epidemiological tools have been developed. In France, a network of 1300 general practitioners, ''Réseau Sentinelles'', working throughout the country, provides real-time clinical data used to evaluate regional and national influenza spreading [1, 2] .", "In France, a network of 1300 general practitioners, ''Réseau Sentinelles'', working throughout the country, provides real-time clinical data used to evaluate regional and national influenza spreading [1, 2] . The criteria used by this network to define clinical influenza-like illness (ILI) are the occurrence of a sudden fever above 39uC with myalgia and respiratory signs. In general no formal viral diagnosis is carried out.", "In general no formal viral diagnosis is carried out. The Groupes Régionaux d'Observation de la Grippe (GROG) is a second French network that surveys the emergence and the spread of the influenza viruses [3, 4] . This network is based on clinical surveillance of acute respiratory infections and laboratory analysis of nasal specimens collected from adults and children by volunteer general practitioners and pediatricians.", "This network is based on clinical surveillance of acute respiratory infections and laboratory analysis of nasal specimens collected from adults and children by volunteer general practitioners and pediatricians. According to the sentinel network's criteria, French health authorities proclaimed that flu epidemic level was reached during the second week of September 2009 (week 37) [5, 6] . On the contrary, data provided by the GROG showed only sporadic H1N1v activity until the last week of October (week 44) [6, 7] .", "On the contrary, data provided by the GROG showed only sporadic H1N1v activity until the last week of October (week 44) [6, 7] . Thus, it became rapidly obvious that a variety of viruses were circulating in the community and that an overestimation of myxovirus infection was at stake [8, 9, 10, 11] . As a better knowledge of the epidemic status was a key feature for national healthcare organization, hospital preparedness, patient management and disease control, unambiguous viral diagnosis appeared critical.", "As a better knowledge of the epidemic status was a key feature for national healthcare organization, hospital preparedness, patient management and disease control, unambiguous viral diagnosis appeared critical. In France, data on viral aetiologies associated with ILI were at best sporadic and correlations with clinical symptoms were often lacking. Extensive molecular assays to screening for respiratory viruses were not available countrywide for routine diagnosis.", "Extensive molecular assays to screening for respiratory viruses were not available countrywide for routine diagnosis. Therefore the epidemiological pattern of respiratory pathogens with overlapping seasonality was poorly known. The aim of the present study was to investigate respiratory pathogens involved in ILI during the early weeks of the 2009-2010 H1N1v diffusion in France (weeks 35 through 44) and describe the associated symptoms in paediatric and adult populations.", "The aim of the present study was to investigate respiratory pathogens involved in ILI during the early weeks of the 2009-2010 H1N1v diffusion in France (weeks 35 through 44) and describe the associated symptoms in paediatric and adult populations. This study was a non-interventional study with no addition to usual proceedures. Biological material and clinical data were obtained only for standard viral diagnostic following physicians' prescriptions (no specific sampling, no modification of the sampling protocol, no supplementary question in the national standardized questionnaire).", "Biological material and clinical data were obtained only for standard viral diagnostic following physicians' prescriptions (no specific sampling, no modification of the sampling protocol, no supplementary question in the national standardized questionnaire). Data analyses were carried out using an anonymized database. According to the French Health Public Law (CSP Art L 1121-1.1), such protocol does not require approval of an ethics committee and is exempted from informed consent application.", "According to the French Health Public Law (CSP Art L 1121-1.1), such protocol does not require approval of an ethics committee and is exempted from informed consent application. In the two academic hospitals, Saint-Louis hospital (SLS) in Paris and Tours hospital (TRS), influenza-like illness (ILI) was defined as a patient suffering from at least one general symptom (fever above 38uC, asthenia, myalgia, shivers or headache) and one respiratory symptom (cough, dyspnoea, rhinitis or pharyngitis), in agreement with the guidelines from the French Institut de Veille Sanitaire (InVS), a governmental institution responsible for surveillance and alert in all domains of public health [12] . Criteria for severe clinical presentation were temperature below 35uC or above 39uC despite antipyretic, cardiac frequency above 120/min, respiratory frequency above 30/min, respiratory distress, systolic arterial pressure below 90 mmHg or altered consciousness.", "Criteria for severe clinical presentation were temperature below 35uC or above 39uC despite antipyretic, cardiac frequency above 120/min, respiratory frequency above 30/min, respiratory distress, systolic arterial pressure below 90 mmHg or altered consciousness. Predisposing factors of critical illness were children younger than one year old, pregnant women, diabetes, chronic pre-existing disease (such as respiratory, cardiovascular, neurologic, renal, hepatic or hematologic diseases) and immunosuppression (associated with HIV infection, organ or hematopoietic stem cells transplantation, receipt of chemotherapy or corticosteroids) [13, 14] . A cluster of suspected influenza infections was defined as at least three possible cases in a week in a closed community (household, school,…) [15] .", "A cluster of suspected influenza infections was defined as at least three possible cases in a week in a closed community (household, school,…) [15] . In the two institutions, the prescription of H1N1v molecular testing was recommended for patients with ILI and with either a severe clinical presentation, an underlying risk factor of complications or a condition which was not improving under antiviral treatment. Investigation of grouped suspected cases was also recommended.", "Investigation of grouped suspected cases was also recommended. From week 35 (last week of August) to 44 (last week of October), 413 endonasal swabs were collected in 3 ml of Universal Transport Medium (Copan Diagnostics Inc, Murrieta, CA) from adults and children seen in emergency rooms for suspected ILI (Table 1 ) and sent to SLS and TRS laboratories for H1N1v detection. The two microbiology laboratories participated in the reference laboratories network for the detection of pandemic influenza H1N1v.", "The two microbiology laboratories participated in the reference laboratories network for the detection of pandemic influenza H1N1v. Clinical data were collected at the time of medical attention and reported by clinicians on a national standardized questionnaire provided by InVS [1, 12] . This questionnaire included the presence or absence of the main general and respiratory symptoms associated with ILI (fever, asthenia, myalgia, shivers, headache, cough, rhinitis, pharyngitis, sudden onset) [12] .", "This questionnaire included the presence or absence of the main general and respiratory symptoms associated with ILI (fever, asthenia, myalgia, shivers, headache, cough, rhinitis, pharyngitis, sudden onset) [12] . Total nucleic acid was extracted from 400 mL of Universal Transport Medium using the EasyMag System (Biomérieux, Marcy l'Etoile, France) in SLS or the EZ1 Advanced XL (Qiagen, Courtaboeuf, France) in TRS, according to the manufacturers' instructions (elution volume: 100 mL in SLS or 90 mL in TRS). Before extraction, 5 ml of an Internal Amplification Control (IAC) which contained an encephalomyocarditis virus (EMC) RNA transcript was added into the sample.", "Before extraction, 5 ml of an Internal Amplification Control (IAC) which contained an encephalomyocarditis virus (EMC) RNA transcript was added into the sample. Pandemic H1N1v infection was diagnosed by real-time reverse transcription-PCR (RT-PCR) assay on a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) according to the protocol of the Centers for Disease Control (CDC) [16] . Other respiratory infections were investigated by a multiplex molecular assay based on the Multiplex Ligation-dependent Probe-Amplification (MLPA) technology (RespiFinder19H, Pathofinder, Maastricht, The Netherlands) that allows the detection and differentiation of 14 respiratory viruses, including influenza virus A (InfA), influenza virus B (InfB), rhinovirus (RHV), parainfluenza viruses 1 to 4 (PIV-1 to PIV-4), human metapneumovirus (hMPV), adenovirus (ADV), respiratory syncytial virus A (RSVA), respiratory syncytial virus B (RSVB) and human coronaviruses 229E, OC43 and NL63 (Cor-229E, Cor-OC43, Cor-NL63) [17] .", "Other respiratory infections were investigated by a multiplex molecular assay based on the Multiplex Ligation-dependent Probe-Amplification (MLPA) technology (RespiFinder19H, Pathofinder, Maastricht, The Netherlands) that allows the detection and differentiation of 14 respiratory viruses, including influenza virus A (InfA), influenza virus B (InfB), rhinovirus (RHV), parainfluenza viruses 1 to 4 (PIV-1 to PIV-4), human metapneumovirus (hMPV), adenovirus (ADV), respiratory syncytial virus A (RSVA), respiratory syncytial virus B (RSVB) and human coronaviruses 229E, OC43 and NL63 (Cor-229E, Cor-OC43, Cor-NL63) [17] . The test allows also the detection of H5N1 influenza A virus and of four bacteria: Chlamydophila pneumoniae (CP), Mycoplasma pneumoniae (MP), Legionella pneumophila (LP) and Bordetella pertussis (BP). The amplified MLPA products were analyzed on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA).", "The amplified MLPA products were analyzed on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA). Fragment sizing analysis was performed with the GeneMarker software (SoftGenetics, LLC, State College, PA). Further testing for H1N1v was carried out with Simplexa TM Influenza A H1N1 (2009) (Focus Diagnostics, Cypress, California) when the CDC real time RT-PCR assay was negative for H1N1 and the RespiFinder19H assay was positive for Influenza A.", "Further testing for H1N1v was carried out with Simplexa TM Influenza A H1N1 (2009) (Focus Diagnostics, Cypress, California) when the CDC real time RT-PCR assay was negative for H1N1 and the RespiFinder19H assay was positive for Influenza A. If this latter assay was negative, H3N2 typing was performed as previously described [18] . Data from our study are summarized as frequencies and percentages for categorical variables.", "Data from our study are summarized as frequencies and percentages for categorical variables. Quantitative variables are presented as medians, 25th and 75th percentiles. To compare those variables according to the viral infection status, Fisher tests \n\nBy using CDC reference assay, H1N1v was detected in 66 samples out of 413 (16.6%), more frequently in SLS (38 samples) than in TRS (28 samples) (p,10 24 ).", "To compare those variables according to the viral infection status, Fisher tests \n\nBy using CDC reference assay, H1N1v was detected in 66 samples out of 413 (16.6%), more frequently in SLS (38 samples) than in TRS (28 samples) (p,10 24 ). Overall, weekly percentage of H1N1v positive endonasal swabs remained under 10% until week 41 and increase significantly after (P Trend ,0.0001) ( Figure 1 ). Rate of H1N1v detection reached 30% in SLS at week 42 and in TRS at week 44.", "Rate of H1N1v detection reached 30% in SLS at week 42 and in TRS at week 44. Overall, this rate was in agreement with results provided by the GROG network, showing an earlier start of H1N1v epidemic in Paris area [7, 19] . All 413 nucleic acid extracts were analyzed using the RespiFinder19H assay ( Figure 2 ).", "All 413 nucleic acid extracts were analyzed using the RespiFinder19H assay ( Figure 2 ). Sixty six patients tested H1N1v positive with CDC real time RT-PCR assay were confirmed with the multiplex assay. Thirteen were also co-infected by one or two other respiratory pathogens (multiple infections) ( Figure 2 ).", "Thirteen were also co-infected by one or two other respiratory pathogens (multiple infections) ( Figure 2 ). Three of the 347 H1N1v negative samples could not be studied with the multiplex assay because they contained RT-PCR inhibitors (no amplification of the internal control). Two hundred and fifteen (62.5%) of the remaining 344 H1N1v negative samples were found positive for at least one respiratory pathogen ( Figure 2 ).", "Two hundred and fifteen (62.5%) of the remaining 344 H1N1v negative samples were found positive for at least one respiratory pathogen ( Figure 2 ). Two hundred and twelve were positive for non influenza pathogens (189 single infections and 23 mixed infections with two, three or four viruses) and three additional single infections by influenza A were identified in SLS, including two by pandemic H1N1v and one by seasonal H3N2, as determined after molecular typing (data not shown). Overall, 68 patients (16.5%) were then positive for H1N1v, one for H3N2 and 212 for non influenza pathogens.", "Overall, 68 patients (16.5%) were then positive for H1N1v, one for H3N2 and 212 for non influenza pathogens. There were 245 single infections (55 with H1N1v and 190 with other respiratory pathogens) and 36 mixed infections (13 with H1N1v and 23 without H1N1v) ( Figure 2 ). Among H1N1v negative single infections, the most prevalent viruses were rhinovirus (62.6%, 119 patients), followed by parainfluenza viruses 1 to 4 (24.2%, 46 patients), adenovirus (5.3%, 10 patients), human coronavirus 229E, OC43 and NL63 (3.2%, 6 patients) and respiratory syncytial virus A and B (2.6%, 5 patients) (Figure 2 ).", "Among H1N1v negative single infections, the most prevalent viruses were rhinovirus (62.6%, 119 patients), followed by parainfluenza viruses 1 to 4 (24.2%, 46 patients), adenovirus (5.3%, 10 patients), human coronavirus 229E, OC43 and NL63 (3.2%, 6 patients) and respiratory syncytial virus A and B (2.6%, 5 patients) (Figure 2 ). In addition, RespiFinder19H assay identified three patients with bacterial infection, two with Mycoplasma pneumoniae (one 25 years old female in SLS and one 39 years old female in TRS) and one with Bordetella pertussis (one 60 years old male in SLS). No single infection by influenza B, hMPV, Chlamydophila pneumoniae or Legionella pneumophila was identified ( Figure 2 To analyze if viral co-infections occurred more frequently for some viruses, we carried out a two by two comparisons, that showed a higher proportion of co-infection only for ADV (p = 0.05).", "No single infection by influenza B, hMPV, Chlamydophila pneumoniae or Legionella pneumophila was identified ( Figure 2 To analyze if viral co-infections occurred more frequently for some viruses, we carried out a two by two comparisons, that showed a higher proportion of co-infection only for ADV (p = 0.05). Non-influenza respiratory viruses presented a different epidemic profile compared to H1N1v. Overall, in both hospitals, weekly rate of non-H1N1v respiratory viruses whether alone or involved in co-infection increased between week 37 and 39 (from 51.4% to 81.3%) and then consistently decreased ( Figure 3 ).", "Overall, in both hospitals, weekly rate of non-H1N1v respiratory viruses whether alone or involved in co-infection increased between week 37 and 39 (from 51.4% to 81.3%) and then consistently decreased ( Figure 3 ). RHV infections that represented nearly half of non-H1N1v viral infections (141 out of 213, 66.2%) were a significant contributing factor. In both hospitals, emergence of H1N1v cases was associated with a rapid decline of RHV rate of infection from 50-60% down to less than 20% with a one to two weeks gap between SLS and TRS.", "In both hospitals, emergence of H1N1v cases was associated with a rapid decline of RHV rate of infection from 50-60% down to less than 20% with a one to two weeks gap between SLS and TRS. Data on age ( In both institutions, 85.5% (106/124) children younger than 15 years of age were infected by at least one respiratory pathogen ( Table 2 ). H1N1v infected patients were not significantly younger than H1N1v non infected patients (27 years old vs. 25 years old, p = 0.80) (Figure 4) .", "H1N1v infected patients were not significantly younger than H1N1v non infected patients (27 years old vs. 25 years old, p = 0.80) (Figure 4) . However, 70.6% (48/68) of H1N1v cases were identified in patients under 40 years old (22 in SLS and 26 in TRS) and no case was observed in patients older than 65 years ( Table 2) . PIV infection occurred in very young patients (median (Figure 4) .", "PIV infection occurred in very young patients (median (Figure 4) . Consequently, PIV and ADV were more frequently detected in the younger population of TRS versus SLS (p,10 24 and p,10 23 respectively). In contrast, although individuals with RHV infection were slightly younger than individuals without (median age = 24 vs. 29 for patients without RHV, p = 0.05) (Figure 4) , influenza-like illness associated with RHV was more frequent in SLS than in TRS (p = 0.012).", "In contrast, although individuals with RHV infection were slightly younger than individuals without (median age = 24 vs. 29 for patients without RHV, p = 0.05) (Figure 4) , influenza-like illness associated with RHV was more frequent in SLS than in TRS (p = 0.012). Finally, patients with viral multiple infection were significantly younger than those with single infection (median, IDR: 4, 2-18.5 vs. 25, 6-43) and rates of mixed infection At the time of medical attention, 383 (92.7%) standardized clinical questionnaires were collected out of 413 patients. Four of them could not be exploited because they were too incomplete.", "Four of them could not be exploited because they were too incomplete. A review of the 379 workable questionnaires showed that 90.8% (344/379) of the patients included in this study fulfilled the criteria of ILI as defined above, and 52.5% had either a severe clinical presentation or an underlying risk factor of complications (45.9%, 174/379), or were in a suspected cluster of grouped cases (6.6%, 25/379). Overall, most patients have fever (93.9%) and cough (86.1%) ( Table 3) .", "Overall, most patients have fever (93.9%) and cough (86.1%) ( Table 3) . Other classical clinical signs associated with ILI such as asthenia, myalgia, shivers, headache, rhinitis or pharyngitis were less frequent. A sudden onset was also described in 59.2% of cases.", "A sudden onset was also described in 59.2% of cases. Only 32.5% of the patients had a temperature above 39uC; the age of these patients ranged from zero to 86 years, with a median age of 32 years and a mean age of 34 years (data not shown). In H1N1v infected patients (including single and multiple infections), the main symptoms were also fever (98.2%) and cough (89.5%) ( We then compared clinical characteristics between patients positive for H1N1v, patients positive for other respiratory pathogens and negative for H1N1v and patients without any detection of respiratory pathogens (as detected with RespiFin-der19H) ( Table 3 ).", "In H1N1v infected patients (including single and multiple infections), the main symptoms were also fever (98.2%) and cough (89.5%) ( We then compared clinical characteristics between patients positive for H1N1v, patients positive for other respiratory pathogens and negative for H1N1v and patients without any detection of respiratory pathogens (as detected with RespiFin-der19H) ( Table 3 ). There was no difference between the three groups except for fever, cough, pharyngitis. However for these latter symptoms, the comparison between patients positive for H1N1v and those positive for other respiratory pathogens or between patients positive for H1N1v and those without any detection of respiratory pathogens, showed no difference except for pharyngitis, which was less frequent in patients positive for H1N1v than in patients positive for other respiratory pathogens ( Table 3) .", "However for these latter symptoms, the comparison between patients positive for H1N1v and those positive for other respiratory pathogens or between patients positive for H1N1v and those without any detection of respiratory pathogens, showed no difference except for pharyngitis, which was less frequent in patients positive for H1N1v than in patients positive for other respiratory pathogens ( Table 3) . As RHV was the most frequent aetiology in ILI, we also compared clinical symptoms observed in patients with a single infection by RHV or by H1N1v (data not shown). There was no difference except that rhinitis and pharyngitis were significantly more frequent in RHV infection (62.7% vs. 34.1% [p = 0.006] and 39.0% vs. 10.0% [p = 0.001], respectively).", "There was no difference except that rhinitis and pharyngitis were significantly more frequent in RHV infection (62.7% vs. 34.1% [p = 0.006] and 39.0% vs. 10.0% [p = 0.001], respectively). Viral multiple infection (including samples with H1N1v) was not associated with a different clinical presentation. Fever and cough were observed in over 90% of the patients (90.6% and 90.3%, respectively), but only 33.3% of these patients had a temperature above 39uC, which was not different from patients with single viral infection (28.6%).", "Fever and cough were observed in over 90% of the patients (90.6% and 90.3%, respectively), but only 33.3% of these patients had a temperature above 39uC, which was not different from patients with single viral infection (28.6%). Our results highlight the high frequency of non-influenza viruses involved in acute respiratory infections during the epidemic period of a flu alert as defined by the Réseau Sentinelles according to ILI definition (a sudden fever above 39uC accompanied by myalgia and respiratory signs). These data extent previous observations in Europe reporting high prevalence of RHV infections before seasonal influenza [4, 20] or in 2009, before H1N1v pandemic influenza [1, 8, 9, 11, 21] .", "These data extent previous observations in Europe reporting high prevalence of RHV infections before seasonal influenza [4, 20] or in 2009, before H1N1v pandemic influenza [1, 8, 9, 11, 21] . We confirm that RHV represent the most frequent aetiology of acute respiratory Table 2 . Age of patients with respiratory samples positive for H1N1v, positive for other respiratory pathogens or negative.", "Age of patients with respiratory samples positive for H1N1v, positive for other respiratory pathogens or negative. infections both in adult and paediatric populations and may represent more than 50% of cases. We show that other viral infections than influenza and RHV may represent up to 30% of aetiologies.", "We show that other viral infections than influenza and RHV may represent up to 30% of aetiologies. We observed differences between the two hospitals, with a higher frequency of parainfluenza and ADV infections in Tours in contrast with a higher frequency of RHV in Paris, likely explained by the higher proportion of paediatric samples collected in Tours. However, despite the distance between the two institutions (about 250 km) and differences between the two populations, both presented similar patterns of high frequency of non-influenza viruses in acute respiratory infections before the flu epidemic wave and a decline when influenza reached epidemic levels.", "However, despite the distance between the two institutions (about 250 km) and differences between the two populations, both presented similar patterns of high frequency of non-influenza viruses in acute respiratory infections before the flu epidemic wave and a decline when influenza reached epidemic levels. In the two cities, high frequencies of RHV were seen at the same level with a likely different evolution speed, with sudden increase and decrease in SLS and more progressive variation in TRS. In both institutions, there was a decrease in the proportion and number of RHV diagnoses roughly in parallel with the increase of influenza diagnoses.", "In both institutions, there was a decrease in the proportion and number of RHV diagnoses roughly in parallel with the increase of influenza diagnoses. Indeed, H1N1v exceeds 20% of positive detection's rate only when RHV dropped under 40%. These data are thus consistent with negative interaction of the two epidemics at the population level.", "These data are thus consistent with negative interaction of the two epidemics at the population level. It was previously hypothesised that RHV epidemic could interfere with the spread of pandemic influenza [20, 21, 22] . Few in vitro data support this hypothesis.", "Few in vitro data support this hypothesis. It has been reported that interferon and other cytokines production by RHV infected cells induced a refractory state to virus infection These data include the three patients whose respiratory samples could not be studied with the multiplex assay because of RT-PCR inhibitors. of neighbouring cells [23] .", "of neighbouring cells [23] . Further work is needed to confirm in vitro and in vivo such negative interactions and if viral interference are really translated to a population level. Analysis of rhinovirus and influenza epidemics in previous years should also help to determine if similar interferences were observed with seasonal influenza and to elaborate modelling and prediction of the spread of influenza according to respiratory viruses' circulation.", "Analysis of rhinovirus and influenza epidemics in previous years should also help to determine if similar interferences were observed with seasonal influenza and to elaborate modelling and prediction of the spread of influenza according to respiratory viruses' circulation. Systematic extensive screening of respiratory viruses at a national level should be implemented for this purpose. Very few RSV infections were observed in contrast to usual epidemiology which was characterized the last four past years by a start of epidemics in weeks 44-45 [1] .", "Very few RSV infections were observed in contrast to usual epidemiology which was characterized the last four past years by a start of epidemics in weeks 44-45 [1] . It has been confirmed by other laboratories and the French InVS that the 2009-10 RSV epidemic was delayed and had a lower impact compared with the previous winter season [1, 24] . Delayed and reduced RSV spread may be due to viral interference between RSV and influenza.", "Delayed and reduced RSV spread may be due to viral interference between RSV and influenza. Another possible explanation is better prevention behaviour about respiratory infections as recommended by a national campaign including recommendations for hands washing after sneezing and the use of mask [1] . Influenza infections were mainly detected in patient under 40 years old and no case was found in patients older than 65.", "Influenza infections were mainly detected in patient under 40 years old and no case was found in patients older than 65. These results corroborate previous data suggesting that past seasonal H1N1 infections or vaccination may give partial crossed protection [10, 13, 25] . We have previously shown that the neutralizing titers against pandemic H1N1v virus correlate significantly with neutralizing titers against a seasonal H1N1 virus, and that the H1N1v pandemic influenza virus neutralizing titer was significantly higher in subjects who had recently been inoculated by a seasonal trivalent influenza vaccine [26] .", "We have previously shown that the neutralizing titers against pandemic H1N1v virus correlate significantly with neutralizing titers against a seasonal H1N1 virus, and that the H1N1v pandemic influenza virus neutralizing titer was significantly higher in subjects who had recently been inoculated by a seasonal trivalent influenza vaccine [26] . Viral co-infections were predominantly seen in paediatric patients, as previously described [4, 27, 28, 29] , both in influenza and non-influenza cases at a similar rate. No evidence of more pronounced respiratory impact was seen in these patients.", "No evidence of more pronounced respiratory impact was seen in these patients. Our results showed the lack of specific clinical signs associated with proven H1N1v infections. Clinical characteristics did not differ between influenza infections or other viral infections. In particular, the proportion of patients with fever above 39uC was not higher in H1N1v positive patients.", "In particular, the proportion of patients with fever above 39uC was not higher in H1N1v positive patients. In addition, the patients without any evidence of respiratory viral infections did not have different symptoms. These patients may have been infected with other virus not included in the multiplex assay (human Bocavirus, coronavirus HKU1) [9, 10, 11] or were seen too late at the time of viral shedding was cleared [30] .", "These patients may have been infected with other virus not included in the multiplex assay (human Bocavirus, coronavirus HKU1) [9, 10, 11] or were seen too late at the time of viral shedding was cleared [30] . However, to determine how specific the symptoms are for influenza would require to assess also the distribution of respiratory pathogens (H1N1v and other respiratory viruses) and related symptoms in patients presented at the emergency departments in SLS and TRS with respiratory syndromes, but not tested for H1N1v. In addition, despite some underlying conditions that were associated with complications not previously observed in seasonal influenza, most illnesses caused by the H1N1v virus were acute and self-limited [13, 31] .", "In addition, despite some underlying conditions that were associated with complications not previously observed in seasonal influenza, most illnesses caused by the H1N1v virus were acute and self-limited [13, 31] . The higher proportion of non influenza viruses reported in ILI in 2009 was thus most likely a consequence of more frequent visits to a doctor for respiratory tract infections than usually observed for fear of the flu pandemic. The general lack of difference in symptoms in the particular context of H1N1v pandemic has therefore to be considered with caution and does not rule out that more significant differences may arise in future influenza epidemics with other influenza viruses.", "The general lack of difference in symptoms in the particular context of H1N1v pandemic has therefore to be considered with caution and does not rule out that more significant differences may arise in future influenza epidemics with other influenza viruses. Our data confirm that it may be virtually impossible to recognize symptoms heralding H1N1v infections and virological data should be helpful along with clinical reports to monitor influenza epidemic [10] . Molecular multiplex detection has recently emerged as a potent diagnostic tool to determine acute respiratory infections' aetiologies [11, 32, 33] .", "Molecular multiplex detection has recently emerged as a potent diagnostic tool to determine acute respiratory infections' aetiologies [11, 32, 33] . These data show that sensitive molecular multiplex detection of respiratory viruses is feasible and efficient for the detection of virus involved in acute respiratory infections and provides insights into their epidemic profile. Our results confirm the performance of RespiFinder19H assay to detecting respiratory viruses in the general population as recently shown in transplant patients with ILI [34] .", "Our results confirm the performance of RespiFinder19H assay to detecting respiratory viruses in the general population as recently shown in transplant patients with ILI [34] . RespiFinder19H confirmed all H1N1 infections detected by the CDC reference assay and was able to identify two additional H1N1 cases suggesting a high sensitivity of this multiplex assay to detect influenza A infections. In conclusion, our results highlight that successive and mixed outbreaks of respiratory viral infections may affect influenza epidemiology and can lead to misinterpret the early development of a flu epidemic.", "In conclusion, our results highlight that successive and mixed outbreaks of respiratory viral infections may affect influenza epidemiology and can lead to misinterpret the early development of a flu epidemic. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management." ]

[ "BACKGROUND: Influenza-like illness (ILI) may be caused by a variety of pathogens. Clinical observations are of little help to recognise myxovirus infection and implement appropriate prevention measures. The limited use of molecular tools underestimates the role of other common pathogens.", "The limited use of molecular tools underestimates the role of other common pathogens. OBJECTIVES: During the early weeks of the 2009–2010 flu pandemic, a clinical and virological survey was conducted in adult and paediatric patients with ILI referred to two French University hospitals in Paris and Tours. Aims were to investigate the different pathogens involved in ILI and describe the associated symptoms.", "Aims were to investigate the different pathogens involved in ILI and describe the associated symptoms. METHODS: H1N1v pandemic influenza diagnosis was performed with real time RT-PCR assay. Other viral aetiologies were investigated by the molecular multiplex assay RespiFinder19®. Clinical data were collected prospectively by physicians using a standard questionnaire.", "Clinical data were collected prospectively by physicians using a standard questionnaire. RESULTS: From week 35 to 44, endonasal swabs were collected in 413 patients. Overall, 68 samples (16.5%) were positive for H1N1v. In 13 of them, other respiratory pathogens were also detected.", "In 13 of them, other respiratory pathogens were also detected. Among H1N1v negative samples, 213 (61.9%) were positive for various respiratory agents, 190 in single infections and 23 in mixed infections. The most prevalent viruses in H1N1v negative single infections were rhinovirus (62.6%), followed by parainfluenza viruses (24.2%) and adenovirus (5.3%).", "The most prevalent viruses in H1N1v negative single infections were rhinovirus (62.6%), followed by parainfluenza viruses (24.2%) and adenovirus (5.3%). 70.6% of H1N1v cases were identified in patients under 40 years and none after 65 years. There was no difference between clinical symptoms observed in patients infected with H1N1v or with other pathogens.", "There was no difference between clinical symptoms observed in patients infected with H1N1v or with other pathogens. CONCLUSION: Our results highlight the high frequency of non-influenza viruses involved in ILI during the pre-epidemic period of a flu alert and the lack of specific clinical signs associated with influenza infections. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management.", "Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management. Text: In order to monitor the spread of influenza and alert health handlers, several epidemiological tools have been developed. In France, a network of 1300 general practitioners, ''Réseau Sentinelles'', working throughout the country, provides real-time clinical data used to evaluate regional and national influenza spreading [1, 2] .", "In France, a network of 1300 general practitioners, ''Réseau Sentinelles'', working throughout the country, provides real-time clinical data used to evaluate regional and national influenza spreading [1, 2] . The criteria used by this network to define clinical influenza-like illness (ILI) are the occurrence of a sudden fever above 39uC with myalgia and respiratory signs. In general no formal viral diagnosis is carried out.", "In general no formal viral diagnosis is carried out. The Groupes Régionaux d'Observation de la Grippe (GROG) is a second French network that surveys the emergence and the spread of the influenza viruses [3, 4] . This network is based on clinical surveillance of acute respiratory infections and laboratory analysis of nasal specimens collected from adults and children by volunteer general practitioners and pediatricians.", "This network is based on clinical surveillance of acute respiratory infections and laboratory analysis of nasal specimens collected from adults and children by volunteer general practitioners and pediatricians. According to the sentinel network's criteria, French health authorities proclaimed that flu epidemic level was reached during the second week of September 2009 (week 37) [5, 6] . On the contrary, data provided by the GROG showed only sporadic H1N1v activity until the last week of October (week 44) [6, 7] .", "On the contrary, data provided by the GROG showed only sporadic H1N1v activity until the last week of October (week 44) [6, 7] . Thus, it became rapidly obvious that a variety of viruses were circulating in the community and that an overestimation of myxovirus infection was at stake [8, 9, 10, 11] . As a better knowledge of the epidemic status was a key feature for national healthcare organization, hospital preparedness, patient management and disease control, unambiguous viral diagnosis appeared critical.", "As a better knowledge of the epidemic status was a key feature for national healthcare organization, hospital preparedness, patient management and disease control, unambiguous viral diagnosis appeared critical. In France, data on viral aetiologies associated with ILI were at best sporadic and correlations with clinical symptoms were often lacking. Extensive molecular assays to screening for respiratory viruses were not available countrywide for routine diagnosis.", "Extensive molecular assays to screening for respiratory viruses were not available countrywide for routine diagnosis. Therefore the epidemiological pattern of respiratory pathogens with overlapping seasonality was poorly known. The aim of the present study was to investigate respiratory pathogens involved in ILI during the early weeks of the 2009-2010 H1N1v diffusion in France (weeks 35 through 44) and describe the associated symptoms in paediatric and adult populations.", "The aim of the present study was to investigate respiratory pathogens involved in ILI during the early weeks of the 2009-2010 H1N1v diffusion in France (weeks 35 through 44) and describe the associated symptoms in paediatric and adult populations. This study was a non-interventional study with no addition to usual proceedures. Biological material and clinical data were obtained only for standard viral diagnostic following physicians' prescriptions (no specific sampling, no modification of the sampling protocol, no supplementary question in the national standardized questionnaire).", "Biological material and clinical data were obtained only for standard viral diagnostic following physicians' prescriptions (no specific sampling, no modification of the sampling protocol, no supplementary question in the national standardized questionnaire). Data analyses were carried out using an anonymized database. According to the French Health Public Law (CSP Art L 1121-1.1), such protocol does not require approval of an ethics committee and is exempted from informed consent application.", "According to the French Health Public Law (CSP Art L 1121-1.1), such protocol does not require approval of an ethics committee and is exempted from informed consent application. In the two academic hospitals, Saint-Louis hospital (SLS) in Paris and Tours hospital (TRS), influenza-like illness (ILI) was defined as a patient suffering from at least one general symptom (fever above 38uC, asthenia, myalgia, shivers or headache) and one respiratory symptom (cough, dyspnoea, rhinitis or pharyngitis), in agreement with the guidelines from the French Institut de Veille Sanitaire (InVS), a governmental institution responsible for surveillance and alert in all domains of public health [12] . Criteria for severe clinical presentation were temperature below 35uC or above 39uC despite antipyretic, cardiac frequency above 120/min, respiratory frequency above 30/min, respiratory distress, systolic arterial pressure below 90 mmHg or altered consciousness.", "Criteria for severe clinical presentation were temperature below 35uC or above 39uC despite antipyretic, cardiac frequency above 120/min, respiratory frequency above 30/min, respiratory distress, systolic arterial pressure below 90 mmHg or altered consciousness. Predisposing factors of critical illness were children younger than one year old, pregnant women, diabetes, chronic pre-existing disease (such as respiratory, cardiovascular, neurologic, renal, hepatic or hematologic diseases) and immunosuppression (associated with HIV infection, organ or hematopoietic stem cells transplantation, receipt of chemotherapy or corticosteroids) [13, 14] . A cluster of suspected influenza infections was defined as at least three possible cases in a week in a closed community (household, school,…) [15] .", "A cluster of suspected influenza infections was defined as at least three possible cases in a week in a closed community (household, school,…) [15] . In the two institutions, the prescription of H1N1v molecular testing was recommended for patients with ILI and with either a severe clinical presentation, an underlying risk factor of complications or a condition which was not improving under antiviral treatment. Investigation of grouped suspected cases was also recommended.", "Investigation of grouped suspected cases was also recommended. From week 35 (last week of August) to 44 (last week of October), 413 endonasal swabs were collected in 3 ml of Universal Transport Medium (Copan Diagnostics Inc, Murrieta, CA) from adults and children seen in emergency rooms for suspected ILI (Table 1 ) and sent to SLS and TRS laboratories for H1N1v detection. The two microbiology laboratories participated in the reference laboratories network for the detection of pandemic influenza H1N1v.", "The two microbiology laboratories participated in the reference laboratories network for the detection of pandemic influenza H1N1v. Clinical data were collected at the time of medical attention and reported by clinicians on a national standardized questionnaire provided by InVS [1, 12] . This questionnaire included the presence or absence of the main general and respiratory symptoms associated with ILI (fever, asthenia, myalgia, shivers, headache, cough, rhinitis, pharyngitis, sudden onset) [12] .", "This questionnaire included the presence or absence of the main general and respiratory symptoms associated with ILI (fever, asthenia, myalgia, shivers, headache, cough, rhinitis, pharyngitis, sudden onset) [12] . Total nucleic acid was extracted from 400 mL of Universal Transport Medium using the EasyMag System (Biomérieux, Marcy l'Etoile, France) in SLS or the EZ1 Advanced XL (Qiagen, Courtaboeuf, France) in TRS, according to the manufacturers' instructions (elution volume: 100 mL in SLS or 90 mL in TRS). Before extraction, 5 ml of an Internal Amplification Control (IAC) which contained an encephalomyocarditis virus (EMC) RNA transcript was added into the sample.", "Before extraction, 5 ml of an Internal Amplification Control (IAC) which contained an encephalomyocarditis virus (EMC) RNA transcript was added into the sample. Pandemic H1N1v infection was diagnosed by real-time reverse transcription-PCR (RT-PCR) assay on a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) according to the protocol of the Centers for Disease Control (CDC) [16] . Other respiratory infections were investigated by a multiplex molecular assay based on the Multiplex Ligation-dependent Probe-Amplification (MLPA) technology (RespiFinder19H, Pathofinder, Maastricht, The Netherlands) that allows the detection and differentiation of 14 respiratory viruses, including influenza virus A (InfA), influenza virus B (InfB), rhinovirus (RHV), parainfluenza viruses 1 to 4 (PIV-1 to PIV-4), human metapneumovirus (hMPV), adenovirus (ADV), respiratory syncytial virus A (RSVA), respiratory syncytial virus B (RSVB) and human coronaviruses 229E, OC43 and NL63 (Cor-229E, Cor-OC43, Cor-NL63) [17] .", "Other respiratory infections were investigated by a multiplex molecular assay based on the Multiplex Ligation-dependent Probe-Amplification (MLPA) technology (RespiFinder19H, Pathofinder, Maastricht, The Netherlands) that allows the detection and differentiation of 14 respiratory viruses, including influenza virus A (InfA), influenza virus B (InfB), rhinovirus (RHV), parainfluenza viruses 1 to 4 (PIV-1 to PIV-4), human metapneumovirus (hMPV), adenovirus (ADV), respiratory syncytial virus A (RSVA), respiratory syncytial virus B (RSVB) and human coronaviruses 229E, OC43 and NL63 (Cor-229E, Cor-OC43, Cor-NL63) [17] . The test allows also the detection of H5N1 influenza A virus and of four bacteria: Chlamydophila pneumoniae (CP), Mycoplasma pneumoniae (MP), Legionella pneumophila (LP) and Bordetella pertussis (BP). The amplified MLPA products were analyzed on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA).", "The amplified MLPA products were analyzed on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA). Fragment sizing analysis was performed with the GeneMarker software (SoftGenetics, LLC, State College, PA). Further testing for H1N1v was carried out with Simplexa TM Influenza A H1N1 (2009) (Focus Diagnostics, Cypress, California) when the CDC real time RT-PCR assay was negative for H1N1 and the RespiFinder19H assay was positive for Influenza A.", "Further testing for H1N1v was carried out with Simplexa TM Influenza A H1N1 (2009) (Focus Diagnostics, Cypress, California) when the CDC real time RT-PCR assay was negative for H1N1 and the RespiFinder19H assay was positive for Influenza A. If this latter assay was negative, H3N2 typing was performed as previously described [18] . Data from our study are summarized as frequencies and percentages for categorical variables.", "Data from our study are summarized as frequencies and percentages for categorical variables. Quantitative variables are presented as medians, 25th and 75th percentiles. To compare those variables according to the viral infection status, Fisher tests \n\nBy using CDC reference assay, H1N1v was detected in 66 samples out of 413 (16.6%), more frequently in SLS (38 samples) than in TRS (28 samples) (p,10 24 ).", "To compare those variables according to the viral infection status, Fisher tests \n\nBy using CDC reference assay, H1N1v was detected in 66 samples out of 413 (16.6%), more frequently in SLS (38 samples) than in TRS (28 samples) (p,10 24 ). Overall, weekly percentage of H1N1v positive endonasal swabs remained under 10% until week 41 and increase significantly after (P Trend ,0.0001) ( Figure 1 ). Rate of H1N1v detection reached 30% in SLS at week 42 and in TRS at week 44.", "Rate of H1N1v detection reached 30% in SLS at week 42 and in TRS at week 44. Overall, this rate was in agreement with results provided by the GROG network, showing an earlier start of H1N1v epidemic in Paris area [7, 19] . All 413 nucleic acid extracts were analyzed using the RespiFinder19H assay ( Figure 2 ).", "All 413 nucleic acid extracts were analyzed using the RespiFinder19H assay ( Figure 2 ). Sixty six patients tested H1N1v positive with CDC real time RT-PCR assay were confirmed with the multiplex assay. Thirteen were also co-infected by one or two other respiratory pathogens (multiple infections) ( Figure 2 ).", "Thirteen were also co-infected by one or two other respiratory pathogens (multiple infections) ( Figure 2 ). Three of the 347 H1N1v negative samples could not be studied with the multiplex assay because they contained RT-PCR inhibitors (no amplification of the internal control). Two hundred and fifteen (62.5%) of the remaining 344 H1N1v negative samples were found positive for at least one respiratory pathogen ( Figure 2 ).", "Two hundred and fifteen (62.5%) of the remaining 344 H1N1v negative samples were found positive for at least one respiratory pathogen ( Figure 2 ). Two hundred and twelve were positive for non influenza pathogens (189 single infections and 23 mixed infections with two, three or four viruses) and three additional single infections by influenza A were identified in SLS, including two by pandemic H1N1v and one by seasonal H3N2, as determined after molecular typing (data not shown). Overall, 68 patients (16.5%) were then positive for H1N1v, one for H3N2 and 212 for non influenza pathogens.", "Overall, 68 patients (16.5%) were then positive for H1N1v, one for H3N2 and 212 for non influenza pathogens. There were 245 single infections (55 with H1N1v and 190 with other respiratory pathogens) and 36 mixed infections (13 with H1N1v and 23 without H1N1v) ( Figure 2 ). Among H1N1v negative single infections, the most prevalent viruses were rhinovirus (62.6%, 119 patients), followed by parainfluenza viruses 1 to 4 (24.2%, 46 patients), adenovirus (5.3%, 10 patients), human coronavirus 229E, OC43 and NL63 (3.2%, 6 patients) and respiratory syncytial virus A and B (2.6%, 5 patients) (Figure 2 ).", "Among H1N1v negative single infections, the most prevalent viruses were rhinovirus (62.6%, 119 patients), followed by parainfluenza viruses 1 to 4 (24.2%, 46 patients), adenovirus (5.3%, 10 patients), human coronavirus 229E, OC43 and NL63 (3.2%, 6 patients) and respiratory syncytial virus A and B (2.6%, 5 patients) (Figure 2 ). In addition, RespiFinder19H assay identified three patients with bacterial infection, two with Mycoplasma pneumoniae (one 25 years old female in SLS and one 39 years old female in TRS) and one with Bordetella pertussis (one 60 years old male in SLS). No single infection by influenza B, hMPV, Chlamydophila pneumoniae or Legionella pneumophila was identified ( Figure 2 To analyze if viral co-infections occurred more frequently for some viruses, we carried out a two by two comparisons, that showed a higher proportion of co-infection only for ADV (p = 0.05).", "No single infection by influenza B, hMPV, Chlamydophila pneumoniae or Legionella pneumophila was identified ( Figure 2 To analyze if viral co-infections occurred more frequently for some viruses, we carried out a two by two comparisons, that showed a higher proportion of co-infection only for ADV (p = 0.05). Non-influenza respiratory viruses presented a different epidemic profile compared to H1N1v. Overall, in both hospitals, weekly rate of non-H1N1v respiratory viruses whether alone or involved in co-infection increased between week 37 and 39 (from 51.4% to 81.3%) and then consistently decreased ( Figure 3 ).", "Overall, in both hospitals, weekly rate of non-H1N1v respiratory viruses whether alone or involved in co-infection increased between week 37 and 39 (from 51.4% to 81.3%) and then consistently decreased ( Figure 3 ). RHV infections that represented nearly half of non-H1N1v viral infections (141 out of 213, 66.2%) were a significant contributing factor. In both hospitals, emergence of H1N1v cases was associated with a rapid decline of RHV rate of infection from 50-60% down to less than 20% with a one to two weeks gap between SLS and TRS.", "In both hospitals, emergence of H1N1v cases was associated with a rapid decline of RHV rate of infection from 50-60% down to less than 20% with a one to two weeks gap between SLS and TRS. Data on age ( In both institutions, 85.5% (106/124) children younger than 15 years of age were infected by at least one respiratory pathogen ( Table 2 ). H1N1v infected patients were not significantly younger than H1N1v non infected patients (27 years old vs. 25 years old, p = 0.80) (Figure 4) .", "H1N1v infected patients were not significantly younger than H1N1v non infected patients (27 years old vs. 25 years old, p = 0.80) (Figure 4) . However, 70.6% (48/68) of H1N1v cases were identified in patients under 40 years old (22 in SLS and 26 in TRS) and no case was observed in patients older than 65 years ( Table 2) . PIV infection occurred in very young patients (median (Figure 4) .", "PIV infection occurred in very young patients (median (Figure 4) . Consequently, PIV and ADV were more frequently detected in the younger population of TRS versus SLS (p,10 24 and p,10 23 respectively). In contrast, although individuals with RHV infection were slightly younger than individuals without (median age = 24 vs. 29 for patients without RHV, p = 0.05) (Figure 4) , influenza-like illness associated with RHV was more frequent in SLS than in TRS (p = 0.012).", "In contrast, although individuals with RHV infection were slightly younger than individuals without (median age = 24 vs. 29 for patients without RHV, p = 0.05) (Figure 4) , influenza-like illness associated with RHV was more frequent in SLS than in TRS (p = 0.012). Finally, patients with viral multiple infection were significantly younger than those with single infection (median, IDR: 4, 2-18.5 vs. 25, 6-43) and rates of mixed infection At the time of medical attention, 383 (92.7%) standardized clinical questionnaires were collected out of 413 patients. Four of them could not be exploited because they were too incomplete.", "Four of them could not be exploited because they were too incomplete. A review of the 379 workable questionnaires showed that 90.8% (344/379) of the patients included in this study fulfilled the criteria of ILI as defined above, and 52.5% had either a severe clinical presentation or an underlying risk factor of complications (45.9%, 174/379), or were in a suspected cluster of grouped cases (6.6%, 25/379). Overall, most patients have fever (93.9%) and cough (86.1%) ( Table 3) .", "Overall, most patients have fever (93.9%) and cough (86.1%) ( Table 3) . Other classical clinical signs associated with ILI such as asthenia, myalgia, shivers, headache, rhinitis or pharyngitis were less frequent. A sudden onset was also described in 59.2% of cases.", "A sudden onset was also described in 59.2% of cases. Only 32.5% of the patients had a temperature above 39uC; the age of these patients ranged from zero to 86 years, with a median age of 32 years and a mean age of 34 years (data not shown). In H1N1v infected patients (including single and multiple infections), the main symptoms were also fever (98.2%) and cough (89.5%) ( We then compared clinical characteristics between patients positive for H1N1v, patients positive for other respiratory pathogens and negative for H1N1v and patients without any detection of respiratory pathogens (as detected with RespiFin-der19H) ( Table 3 ).", "In H1N1v infected patients (including single and multiple infections), the main symptoms were also fever (98.2%) and cough (89.5%) ( We then compared clinical characteristics between patients positive for H1N1v, patients positive for other respiratory pathogens and negative for H1N1v and patients without any detection of respiratory pathogens (as detected with RespiFin-der19H) ( Table 3 ). There was no difference between the three groups except for fever, cough, pharyngitis. However for these latter symptoms, the comparison between patients positive for H1N1v and those positive for other respiratory pathogens or between patients positive for H1N1v and those without any detection of respiratory pathogens, showed no difference except for pharyngitis, which was less frequent in patients positive for H1N1v than in patients positive for other respiratory pathogens ( Table 3) .", "However for these latter symptoms, the comparison between patients positive for H1N1v and those positive for other respiratory pathogens or between patients positive for H1N1v and those without any detection of respiratory pathogens, showed no difference except for pharyngitis, which was less frequent in patients positive for H1N1v than in patients positive for other respiratory pathogens ( Table 3) . As RHV was the most frequent aetiology in ILI, we also compared clinical symptoms observed in patients with a single infection by RHV or by H1N1v (data not shown). There was no difference except that rhinitis and pharyngitis were significantly more frequent in RHV infection (62.7% vs. 34.1% [p = 0.006] and 39.0% vs. 10.0% [p = 0.001], respectively).", "There was no difference except that rhinitis and pharyngitis were significantly more frequent in RHV infection (62.7% vs. 34.1% [p = 0.006] and 39.0% vs. 10.0% [p = 0.001], respectively). Viral multiple infection (including samples with H1N1v) was not associated with a different clinical presentation. Fever and cough were observed in over 90% of the patients (90.6% and 90.3%, respectively), but only 33.3% of these patients had a temperature above 39uC, which was not different from patients with single viral infection (28.6%).", "Fever and cough were observed in over 90% of the patients (90.6% and 90.3%, respectively), but only 33.3% of these patients had a temperature above 39uC, which was not different from patients with single viral infection (28.6%). Our results highlight the high frequency of non-influenza viruses involved in acute respiratory infections during the epidemic period of a flu alert as defined by the Réseau Sentinelles according to ILI definition (a sudden fever above 39uC accompanied by myalgia and respiratory signs). These data extent previous observations in Europe reporting high prevalence of RHV infections before seasonal influenza [4, 20] or in 2009, before H1N1v pandemic influenza [1, 8, 9, 11, 21] .", "These data extent previous observations in Europe reporting high prevalence of RHV infections before seasonal influenza [4, 20] or in 2009, before H1N1v pandemic influenza [1, 8, 9, 11, 21] . We confirm that RHV represent the most frequent aetiology of acute respiratory Table 2 . Age of patients with respiratory samples positive for H1N1v, positive for other respiratory pathogens or negative.", "Age of patients with respiratory samples positive for H1N1v, positive for other respiratory pathogens or negative. infections both in adult and paediatric populations and may represent more than 50% of cases. We show that other viral infections than influenza and RHV may represent up to 30% of aetiologies.", "We show that other viral infections than influenza and RHV may represent up to 30% of aetiologies. We observed differences between the two hospitals, with a higher frequency of parainfluenza and ADV infections in Tours in contrast with a higher frequency of RHV in Paris, likely explained by the higher proportion of paediatric samples collected in Tours. However, despite the distance between the two institutions (about 250 km) and differences between the two populations, both presented similar patterns of high frequency of non-influenza viruses in acute respiratory infections before the flu epidemic wave and a decline when influenza reached epidemic levels.", "However, despite the distance between the two institutions (about 250 km) and differences between the two populations, both presented similar patterns of high frequency of non-influenza viruses in acute respiratory infections before the flu epidemic wave and a decline when influenza reached epidemic levels. In the two cities, high frequencies of RHV were seen at the same level with a likely different evolution speed, with sudden increase and decrease in SLS and more progressive variation in TRS. In both institutions, there was a decrease in the proportion and number of RHV diagnoses roughly in parallel with the increase of influenza diagnoses.", "In both institutions, there was a decrease in the proportion and number of RHV diagnoses roughly in parallel with the increase of influenza diagnoses. Indeed, H1N1v exceeds 20% of positive detection's rate only when RHV dropped under 40%. These data are thus consistent with negative interaction of the two epidemics at the population level.", "These data are thus consistent with negative interaction of the two epidemics at the population level. It was previously hypothesised that RHV epidemic could interfere with the spread of pandemic influenza [20, 21, 22] . Few in vitro data support this hypothesis.", "Few in vitro data support this hypothesis. It has been reported that interferon and other cytokines production by RHV infected cells induced a refractory state to virus infection These data include the three patients whose respiratory samples could not be studied with the multiplex assay because of RT-PCR inhibitors. of neighbouring cells [23] .", "of neighbouring cells [23] . Further work is needed to confirm in vitro and in vivo such negative interactions and if viral interference are really translated to a population level. Analysis of rhinovirus and influenza epidemics in previous years should also help to determine if similar interferences were observed with seasonal influenza and to elaborate modelling and prediction of the spread of influenza according to respiratory viruses' circulation.", "Analysis of rhinovirus and influenza epidemics in previous years should also help to determine if similar interferences were observed with seasonal influenza and to elaborate modelling and prediction of the spread of influenza according to respiratory viruses' circulation. Systematic extensive screening of respiratory viruses at a national level should be implemented for this purpose. Very few RSV infections were observed in contrast to usual epidemiology which was characterized the last four past years by a start of epidemics in weeks 44-45 [1] .", "Very few RSV infections were observed in contrast to usual epidemiology which was characterized the last four past years by a start of epidemics in weeks 44-45 [1] . It has been confirmed by other laboratories and the French InVS that the 2009-10 RSV epidemic was delayed and had a lower impact compared with the previous winter season [1, 24] . Delayed and reduced RSV spread may be due to viral interference between RSV and influenza.", "Delayed and reduced RSV spread may be due to viral interference between RSV and influenza. Another possible explanation is better prevention behaviour about respiratory infections as recommended by a national campaign including recommendations for hands washing after sneezing and the use of mask [1] . Influenza infections were mainly detected in patient under 40 years old and no case was found in patients older than 65.", "Influenza infections were mainly detected in patient under 40 years old and no case was found in patients older than 65. These results corroborate previous data suggesting that past seasonal H1N1 infections or vaccination may give partial crossed protection [10, 13, 25] . We have previously shown that the neutralizing titers against pandemic H1N1v virus correlate significantly with neutralizing titers against a seasonal H1N1 virus, and that the H1N1v pandemic influenza virus neutralizing titer was significantly higher in subjects who had recently been inoculated by a seasonal trivalent influenza vaccine [26] .", "We have previously shown that the neutralizing titers against pandemic H1N1v virus correlate significantly with neutralizing titers against a seasonal H1N1 virus, and that the H1N1v pandemic influenza virus neutralizing titer was significantly higher in subjects who had recently been inoculated by a seasonal trivalent influenza vaccine [26] . Viral co-infections were predominantly seen in paediatric patients, as previously described [4, 27, 28, 29] , both in influenza and non-influenza cases at a similar rate. No evidence of more pronounced respiratory impact was seen in these patients.", "No evidence of more pronounced respiratory impact was seen in these patients. Our results showed the lack of specific clinical signs associated with proven H1N1v infections. Clinical characteristics did not differ between influenza infections or other viral infections. In particular, the proportion of patients with fever above 39uC was not higher in H1N1v positive patients.", "In particular, the proportion of patients with fever above 39uC was not higher in H1N1v positive patients. In addition, the patients without any evidence of respiratory viral infections did not have different symptoms. These patients may have been infected with other virus not included in the multiplex assay (human Bocavirus, coronavirus HKU1) [9, 10, 11] or were seen too late at the time of viral shedding was cleared [30] .", "These patients may have been infected with other virus not included in the multiplex assay (human Bocavirus, coronavirus HKU1) [9, 10, 11] or were seen too late at the time of viral shedding was cleared [30] . However, to determine how specific the symptoms are for influenza would require to assess also the distribution of respiratory pathogens (H1N1v and other respiratory viruses) and related symptoms in patients presented at the emergency departments in SLS and TRS with respiratory syndromes, but not tested for H1N1v. In addition, despite some underlying conditions that were associated with complications not previously observed in seasonal influenza, most illnesses caused by the H1N1v virus were acute and self-limited [13, 31] .", "In addition, despite some underlying conditions that were associated with complications not previously observed in seasonal influenza, most illnesses caused by the H1N1v virus were acute and self-limited [13, 31] . The higher proportion of non influenza viruses reported in ILI in 2009 was thus most likely a consequence of more frequent visits to a doctor for respiratory tract infections than usually observed for fear of the flu pandemic. The general lack of difference in symptoms in the particular context of H1N1v pandemic has therefore to be considered with caution and does not rule out that more significant differences may arise in future influenza epidemics with other influenza viruses.", "The general lack of difference in symptoms in the particular context of H1N1v pandemic has therefore to be considered with caution and does not rule out that more significant differences may arise in future influenza epidemics with other influenza viruses. Our data confirm that it may be virtually impossible to recognize symptoms heralding H1N1v infections and virological data should be helpful along with clinical reports to monitor influenza epidemic [10] . Molecular multiplex detection has recently emerged as a potent diagnostic tool to determine acute respiratory infections' aetiologies [11, 32, 33] .", "Molecular multiplex detection has recently emerged as a potent diagnostic tool to determine acute respiratory infections' aetiologies [11, 32, 33] . These data show that sensitive molecular multiplex detection of respiratory viruses is feasible and efficient for the detection of virus involved in acute respiratory infections and provides insights into their epidemic profile. Our results confirm the performance of RespiFinder19H assay to detecting respiratory viruses in the general population as recently shown in transplant patients with ILI [34] .", "Our results confirm the performance of RespiFinder19H assay to detecting respiratory viruses in the general population as recently shown in transplant patients with ILI [34] . RespiFinder19H confirmed all H1N1 infections detected by the CDC reference assay and was able to identify two additional H1N1 cases suggesting a high sensitivity of this multiplex assay to detect influenza A infections. In conclusion, our results highlight that successive and mixed outbreaks of respiratory viral infections may affect influenza epidemiology and can lead to misinterpret the early development of a flu epidemic.", "In conclusion, our results highlight that successive and mixed outbreaks of respiratory viral infections may affect influenza epidemiology and can lead to misinterpret the early development of a flu epidemic. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management." ]

[ "There is increasing concern about the impact of veterinary drugs and livestock pathogens as factors damaging wildlife health, especially of threatened avian scavengers feeding upon medicated livestock carcasses. We conducted a comprehensive study of failed eggs and dead nestlings in bearded vultures (Gypaetus barbatus) to attempt to elucidate the proximate causes of breeding failure behind the recent decline in productivity in the Spanish Pyrenees. We found high concentrations of multiple veterinary drugs, primarily fluoroquinolones, in most failed eggs and nestlings, associated with multiple internal organ damage and livestock pathogens causing disease, especially septicaemia by swine pathogens and infectious bursal disease.", "We found high concentrations of multiple veterinary drugs, primarily fluoroquinolones, in most failed eggs and nestlings, associated with multiple internal organ damage and livestock pathogens causing disease, especially septicaemia by swine pathogens and infectious bursal disease. The combined impact of drugs and disease as stochastic factors may result in potentially devastating effects exacerbating an already high risk of extinction and should be considered in current conservation programs for bearded vultures and other scavenger species, especially in regards to dangerous veterinary drugs and highly pathogenic poultry viruses. Text: Environmental pollutants are increasingly documented as a driver of wildlife endangerment due to their roles in organ damage, hormonal disruption and alteration of the immune system [1, 2] .", "Text: Environmental pollutants are increasingly documented as a driver of wildlife endangerment due to their roles in organ damage, hormonal disruption and alteration of the immune system [1, 2] . Disease may also facilitate endangerment and extinction at global and local scales, especially when pathogens interact with other drivers such as pollutants [3] . There is increasing concern about the impact of veterinary drugs and livestock pathogens as factors damaging wildlife health [4] [5] [6] , and even causing declines approaching extinction [7] .", "There is increasing concern about the impact of veterinary drugs and livestock pathogens as factors damaging wildlife health [4] [5] [6] , and even causing declines approaching extinction [7] . These threats may be especially detrimental to wildlife as they increasingly concur and interact as a consequence of the elimination of livestock residues containing veterinary pharmaceuticals and resistant pathogens due to growing intensive livestock operations worldwide [6, 8, 9] . In particular, the ingestion of antimicrobials, primarily fluoroquinolones, has been recently related to immunodepression-mediated acquisition of opportunistic pathogens and disease, as well as to organ damage in nestling vultures [6, 10, 11] .", "In particular, the ingestion of antimicrobials, primarily fluoroquinolones, has been recently related to immunodepression-mediated acquisition of opportunistic pathogens and disease, as well as to organ damage in nestling vultures [6, 10, 11] . Fluoroquinolone residues have also been found in avian scavenger eggs and are associated with severe alterations in the development of embryo cartilage and bones that could preclude embryo movement and subsequently normal development, pre-hatch position and successful hatching [12] . Therefore, antimicrobials and other drugs may negatively affect embryo and nestling health with potentially devastating consequences on breeding success and conservation of vultures and other threatened avian scavengers.", "Therefore, antimicrobials and other drugs may negatively affect embryo and nestling health with potentially devastating consequences on breeding success and conservation of vultures and other threatened avian scavengers. The bearded vulture (Gypaetus barbatus) is one of the most endangered birds in Europe, with a main stronghold in the Pyrenees. Increasing declines in productivity (average number of fledglings raised per territorial pair) have recently been reported in the Spanish Pyrenees associated with habitat saturation processes [13, 14] .", "Increasing declines in productivity (average number of fledglings raised per territorial pair) have recently been reported in the Spanish Pyrenees associated with habitat saturation processes [13, 14] . Given that bearded vultures may raise only one fledgling per breeding attempt, this productivity decline should be linked to increasing breeding failure when the proportion of territorial pairs that are breeding does not greatly vary with time [15] . The proximate mechanisms by which density can affect productivity have been investigated, including habitat heterogeneity, with progressively poorer territories being used, territory shrinkage and interference with breeders and floaters [13] .", "The proximate mechanisms by which density can affect productivity have been investigated, including habitat heterogeneity, with progressively poorer territories being used, territory shrinkage and interference with breeders and floaters [13] . However, the proximate causes of breeding failure are poorly known despite the long-term interests in the conservation of this species [16] . To evaluate these causes, the examination of failed eggs and dead nestlings is imperative, including the study of the presence and impact of injury, developmental problems, poor nutritional condition, pollutants, organ damage, pathogens causing disease, etc.", "To evaluate these causes, the examination of failed eggs and dead nestlings is imperative, including the study of the presence and impact of injury, developmental problems, poor nutritional condition, pollutants, organ damage, pathogens causing disease, etc. in order to determine the most likely cause of breeding failure. Here, we conducted a comprehensive study of failed eggs and dead nestling bearded vultures collected during recent years in the Pyrenees.", "Here, we conducted a comprehensive study of failed eggs and dead nestling bearded vultures collected during recent years in the Pyrenees. Both the productivity and survival rates of adults and young birds have reached the lowest values since the bovine spongiform encephalopathy (BSE) crisis [13, 14, 17] . This temporal decline could be related to illegal poisoning [17] and recent changes in the abundance, distribution and quality of carrion available to avian scavengers as a consequence of EU regulations derived from the BSE crisis [6, [18] [19] [20] .", "This temporal decline could be related to illegal poisoning [17] and recent changes in the abundance, distribution and quality of carrion available to avian scavengers as a consequence of EU regulations derived from the BSE crisis [6, [18] [19] [20] . In particular, the BSE crisis caused the lack or scarcity of unstabled livestock available to scavengers and their subsequent increase in the consumption of carrion from stabled livestock, which is intensively medicated [21] . Therefore, we specifically focused on determining whether breeding failure in bearded vultures is related to the ingestion of veterinary drugs from stabled livestock carrion, as documented in other avian scavenger species [12] .", "Therefore, we specifically focused on determining whether breeding failure in bearded vultures is related to the ingestion of veterinary drugs from stabled livestock carrion, as documented in other avian scavenger species [12] . We also assessed the potential effects of veterinary drugs on embryo damage and immunodepression increasing the probability of acquisition and proliferation of pathogens causing fatal disease [6, [10] [11] [12] 21] . Because veterinary drugs should be exclusively acquired from the ingestion of carrion from livestock medicated to combat disease, we predict that their presence should be associated with that of pathogens acquired from the same livestock, especially poultry pathogens more likely transmitted between avian species [22] .", "Because veterinary drugs should be exclusively acquired from the ingestion of carrion from livestock medicated to combat disease, we predict that their presence should be associated with that of pathogens acquired from the same livestock, especially poultry pathogens more likely transmitted between avian species [22] . Alternatively, if the temporal decline in productivity was primarily associated with breeding failure due to the effects of habitat saturation processes [13, 17] , we should expect egg and nestling mortality to be directly related to developmental and nutritional problems indicating progressively lower quality territories (e.g. embryo emaciation, nestling starvation) and interference by both conspecifics and heterospecifics (e.g.", "embryo emaciation, nestling starvation) and interference by both conspecifics and heterospecifics (e.g. incubation failure, injury due to predation attempts or disturbance). Failed eggs (n = 5) and dead nestlings (n = 4) were collected from bearded vulture nests located in the Spanish Pyrenees between 2005 and 2008.", "Failed eggs (n = 5) and dead nestlings (n = 4) were collected from bearded vulture nests located in the Spanish Pyrenees between 2005 and 2008. The study of this material did not require of the approval of an ethics committee because it was collected after breeding failure (egg or nestling death) was confirmed in the field. Three of the specimens (two nestlings and one egg) were collected in 2005, 2007 and 2008 from a particular territory.", "Three of the specimens (two nestlings and one egg) were collected in 2005, 2007 and 2008 from a particular territory. Eggs and nestlings were collected after breeding failure and frozen. Necropsies were performed on all specimens according to standard protocols [12] . The age of embryos and nestlings were estimated according to size and development.", "The age of embryos and nestlings were estimated according to size and development. Samples of liver, kidney, spleen, large and small intestines, lungs, brain, lymphoid organs (thymus, bursa of Fabricius, Peyer's patches) and knee joints were fixed in 10% buffered formalin, sectioned at 4 mm and stained for histopathological analysis [10, 12] . Liver (dead nestlings and failed embryos) and yolk (failed embryos) were used for the determination of the presence of veterinary drugs, including fluoroquinolones (enrofloxacin and ciprofloxacin), other antimicrobials (amoxicillin and oxytetracycline), non-steroideal anti-inflamatories (NSAIDs) such as diclofenac, flunixin meglumine, ketoprofen, ibuprofen, meloxicam, sodium salicylate, acetaminophen, and antiparasitics (metronidazole, diclazuril, fenbendazole, ivermectin) as described previously [12] .", "Liver (dead nestlings and failed embryos) and yolk (failed embryos) were used for the determination of the presence of veterinary drugs, including fluoroquinolones (enrofloxacin and ciprofloxacin), other antimicrobials (amoxicillin and oxytetracycline), non-steroideal anti-inflamatories (NSAIDs) such as diclofenac, flunixin meglumine, ketoprofen, ibuprofen, meloxicam, sodium salicylate, acetaminophen, and antiparasitics (metronidazole, diclazuril, fenbendazole, ivermectin) as described previously [12] . The limits of quantification, percentage recoveries, and interand intra-assay reproducibility were adequate [10, 12] . Other contaminants potentially affecting eggs and embryos were determined in liver, including heavy metals (Cd, Zn, Pb and Hg), following Blanco et al.", "Other contaminants potentially affecting eggs and embryos were determined in liver, including heavy metals (Cd, Zn, Pb and Hg), following Blanco et al. [23] , dithiocarbamate thiram, disulfuram, polybrominated diphenyl ethers, organochlorines and brominated flame retardants, following Lemus et al. [12] and carbamate and organophosphate pesticides (carbofuran, aldicarb and fenthion) following Elliot et al.", "[12] and carbamate and organophosphate pesticides (carbofuran, aldicarb and fenthion) following Elliot et al. [24] . We measured brain cholinesterase activity to assess early exposure to anticholinesterase pesticides [25] .", "We measured brain cholinesterase activity to assess early exposure to anticholinesterase pesticides [25] . Potential contamination was assessed by comparison with levels from apparently normal wild birds of other species [26] in the absence of basal levels for bearded vultures. Determination of bacterial and fungal pathogens were conducted by sampling oropharynx, lung, liver, kidney, spleen, and intestine with sterile swabs and cultured using standard microbiology protocols [10, 12, 27, 28] .", "Determination of bacterial and fungal pathogens were conducted by sampling oropharynx, lung, liver, kidney, spleen, and intestine with sterile swabs and cultured using standard microbiology protocols [10, 12, 27, 28] . Salmonella serotypes and phage types were determined in the Spanish Reference Laboratory (Laboratorio Central Veterinario, Algete, Madrid). For confirmation of the identification of the alpha hemolytic Streptococcus pneumoniae we used a specific identification test (Accuprobe, Salem, MA) based on the detection of specific ribosomal RNA sequences.", "For confirmation of the identification of the alpha hemolytic Streptococcus pneumoniae we used a specific identification test (Accuprobe, Salem, MA) based on the detection of specific ribosomal RNA sequences. Samples of lesions found in internal organs and tissues during necropsies were taken with sterile swabs and cultured using the same standard microbiology protocols. In addition, we determined the presence of selected avian pathogens, including bacterial, viral, fungal, and protozoan pathogens by means of PCR-based methods (see Table S1 for details).", "In addition, we determined the presence of selected avian pathogens, including bacterial, viral, fungal, and protozoan pathogens by means of PCR-based methods (see Table S1 for details). The presence of Chlamyophila psittaci and Mycoplasma sp. in blood was determined as described previously [29, 30] .", "in blood was determined as described previously [29, 30] . The presence of poxvirus, the paramyxovirus causing Newcastle disease, the serotypes H5, H7 and H9 of avian influenza, falcon adenovirus, circovirus, herpesvirus, polyomavirus, reovirus and West Nile virus were determined following the PCR-based methods available in the literature [31] [32] [33] [34] [35] [36] [37] [38] [39] . We also searched for helminths and protozoans in the gastrointestinal tract by macroscopic and microscopic observations using standard protocols [40] .", "We also searched for helminths and protozoans in the gastrointestinal tract by macroscopic and microscopic observations using standard protocols [40] . Specific immunocytochemical procedures were used for detection of mielodepressive virus, including the alphaherpesvirus causing Marek disease [41] in kidney and bursa of Fabricius, the gyrovirus causing infectious chicken anaemia [42] in thymus and bone marrow, the birnavirus causing infectious bursal disease (IBD, [43] ) in bursa of Fabricius, and the coronavirus causing chicken infectious bronchitis in kidney [44] . In addition, we conducted a specific immunocytochemical procedure for West Nile virus antigen detection [45] in brain, spinal medulla, thymus and thyroid.", "In addition, we conducted a specific immunocytochemical procedure for West Nile virus antigen detection [45] in brain, spinal medulla, thymus and thyroid. All immunohistochemistry analyses were conducted at the Department of Veterinary Anatomy, Veterinary Faculty, Universidad Complutense de Madrid, Spain and at the Pathology Department of the Veterinary Faculty, University of Utrecht, The Netherlands. The presence of these viruses was also determined by PCR-based methods [43, [46] [47] [48] .", "The presence of these viruses was also determined by PCR-based methods [43, [46] [47] [48] . All dead nestlings and three of five unhatched embryos showed two to six different veterinary drugs in liver (nestlings) and egg yolk (embryos). In addition, the two embryos with fluoroquinolones in the yolk also had them in the liver (Table 1) .", "In addition, the two embryos with fluoroquinolones in the yolk also had them in the liver (Table 1) . Fluoroquinolones were the most prevalent drugs and showed the highest concentrations (Table 1) . Other drugs such as NSAIDs and antiparasitics were found in most nestlings at variable concentrations, but in no eggs (Table 1) .", "Other drugs such as NSAIDs and antiparasitics were found in most nestlings at variable concentrations, but in no eggs (Table 1) . Other toxic compounds were detected in lower prevalence and concentrations (see Table 1 for those more relevant values; all insecticides were found at concentrations ,0.001 ppb), which was further supported by basal levels of brain cholinesterase (Table 1) . Dead embryos and nestlings showed a moderate to good nutritional state.", "Dead embryos and nestlings showed a moderate to good nutritional state. Major histopathological lesions were primarily located in the kidney, including glomerulonephritis and/or glomerulonephrosis present in all individuals with fluoroquinolones, but not in those without drugs (Table 1 ). All individuals with fluoroquinolones also showed joint lesions, including arthritis and/ or arthrosis of the long bone articulations, as well as massive osseous stroma of the spongeous bones.", "All individuals with fluoroquinolones also showed joint lesions, including arthritis and/ or arthrosis of the long bone articulations, as well as massive osseous stroma of the spongeous bones. The fungi Candida albicans was isolated from the oral cavity of five individuals. All individuals showed non-specific mixedbacterial flora. Enterotoxigenic Escherichia coli and Salmonella spp.", "All individuals showed non-specific mixedbacterial flora. Enterotoxigenic Escherichia coli and Salmonella spp. were isolated in four cases ( Table 1 (cont.) *Samples from the same territory in different years. 1 Veterinary drugs.", "*Samples from the same territory in different years. 1 Veterinary drugs. EN: enrofloxacin (mg/g), CI: ciprofloxacin (mg/g), OX: oxytetracyclin (mg/g), FL: flunixin meglumine (mg/g), AS: sodium salicylate (ng/g), IV: Ivermectin (mg/g). 2 Other toxicants.", "2 Other toxicants. OR: organochlorines (ng/g), Pb: lead (ng/g). 12. z29 (three cases, see Table 1 ).", "12. z29 (three cases, see Table 1 ). One individual showed infection by Salmonella enterica enteritidis (see above) and enterotoxigenic Escherichia coli O86 in all examined organs (septicaemia) except brain, which rejected the possibility of post-mortem contamination. Pasteurella multocida was isolated in a single individual that also showed enterotoxigenic Escherichia coli O86 (Table 1) ; all of these individuals contained fluoroquinolones.", "Pasteurella multocida was isolated in a single individual that also showed enterotoxigenic Escherichia coli O86 (Table 1) ; all of these individuals contained fluoroquinolones. One of the failed embryos without veterinary drugs showed suppurative myocarditis, multiple microabscesses in head muscles, suppurative leptomeningitis, as well as lower jaw gangrenous inflammation with loss of the osseous stroma due to a mixed infection with Streptococcus suis and Streptococcus pneumoniae in brain, meninges and neck muscles; this embryo also showed infection by chicken infectious bronchitis ( Table 1 ). Both immunocytochemistry for the detection of poultry viruses and PCR pathogen survey were positive to IBDV in six individuals with fluoroquinolones (Table 1) .", "Both immunocytochemistry for the detection of poultry viruses and PCR pathogen survey were positive to IBDV in six individuals with fluoroquinolones (Table 1) . Immunocytochemical procedures failed to detect West Nile virus antigens in individuals in which PCR for this virus had been positive. Parasitology was negative for all helminths, helminth eggs and protozoans.", "Parasitology was negative for all helminths, helminth eggs and protozoans. We found multiple veterinary drugs, primarily fluoroquinolones, in most failed eggs and dead nestling bearded vultures from the Pyrenees. They also showed multiple internal organ damage and pathogens potentially acquired from medicated livestock carrion, especially viruses often infecting poultry.", "They also showed multiple internal organ damage and pathogens potentially acquired from medicated livestock carrion, especially viruses often infecting poultry. Recorded drug concentrations were among the highest reported in avian scavengers [6, [10] [11] [12] 21] . NSAIDs and antiparasitics were found in lower prevalence than fluoroquinolones, but at higher concentrations than those found in other avian scavengers, especially for flunixin meglumine and sodium salicylate [6, 12, 21] .", "NSAIDs and antiparasitics were found in lower prevalence than fluoroquinolones, but at higher concentrations than those found in other avian scavengers, especially for flunixin meglumine and sodium salicylate [6, 12, 21] . On the contrary, we found no sterile eggs, poor nutritional conditions or injury in any failed embryo or nestling. Other pollutants were found in low prevalence and concentrations posing low risk to embryo and nestling health.", "Other pollutants were found in low prevalence and concentrations posing low risk to embryo and nestling health. Fluoroquinolones may cause generalized direct developmental damage precluding embryo hatching, physiological alterations due to their impact on liver and kidney and immunodepression reducing resistance to opportunistic pathogens [6, [10] [11] [12] 21] . These pathogens may be acquired at the same time that drugs used to treat diseased livestock are ingested, as indicated by their high prevalence in embryos and nestlings.", "These pathogens may be acquired at the same time that drugs used to treat diseased livestock are ingested, as indicated by their high prevalence in embryos and nestlings. Therefore, despite the relatively small sample size resulting from low abundance, endangerment and logistic difficulties in reaching nests in this species, the results provide evidence of a combined impact of veterinary drugs and livestock disease as the primary cause of breeding failure in the sampled individuals. The presence of West Nile virus is not likely to be associated with nestling disease or mortality because the lack of lesions in target tissues and viral antigen particles in the immunohistochemistry study.", "The presence of West Nile virus is not likely to be associated with nestling disease or mortality because the lack of lesions in target tissues and viral antigen particles in the immunohistochemistry study. Fatal septicaemia caused by Streptococcus suis, one of the most important swine pathogens worldwide [49] , in combination with septicaemia from Streptococcus pneumoniae and infection by chicken infectious bronchitis virus were found in a single embryo. This concentration of livestock pathogens has not been reported before and, to our knowledge, this is the first report of the three pathogens causing disease in a wild bird.", "This concentration of livestock pathogens has not been reported before and, to our knowledge, this is the first report of the three pathogens causing disease in a wild bird. Other pathogens recorded in embryos and nestlings, including Salmonella serotypes and phages typical of livestock [50] , and enterotoxigenic Escherichia coli O86 causing septicaemia, were potentially transmitted by consumption of carcasses of infected poultry and other livestock [22, 27, 28] . In addition, we found that the IBD virus infected most individuals alone or together with other pathogens also potentially acquired from livestock carrion.", "In addition, we found that the IBD virus infected most individuals alone or together with other pathogens also potentially acquired from livestock carrion. This virus causes a highly contagious immunosuppressive bursal disease in poultry [51] and may be transmitted to wildlife in contact with poultry waste or by ingestion of carcasses [22, 52] . Nestlings are especially susceptible to IBD because of the primary role of bursa of Fabricius in immune function development at this age.", "Nestlings are especially susceptible to IBD because of the primary role of bursa of Fabricius in immune function development at this age. In fact, immunosuppression due to IBD was indicated by the inflammation, necrosis and loss of lymphocytes in the bursa of Fabricius together with the presence of viral antigens recorded by means of immunocytochemical procedures. The potential impact of highly pathogenic and contagious poultry viruses has been previously recognized as a threat to wildlife health due to the increasing contact of wildlife with livestock operations in general, and poultry farms and their residues in particular, in natural areas worldwide [52] [53] [54] [55] .", "The potential impact of highly pathogenic and contagious poultry viruses has been previously recognized as a threat to wildlife health due to the increasing contact of wildlife with livestock operations in general, and poultry farms and their residues in particular, in natural areas worldwide [52] [53] [54] [55] . However, damage from IBD virus on the bursa of Fabricius represents, to our knowledge, the first evidence of clinical disease compatible with death caused by this poultry virus in wildlife. The presence of IBD has been not previously recorded in embryos of wild birds, probably because vertical transmission has been ruled out in poultry and, as consequence, it has probably not been evaluated in other species until now.", "The presence of IBD has been not previously recorded in embryos of wild birds, probably because vertical transmission has been ruled out in poultry and, as consequence, it has probably not been evaluated in other species until now. This striking and concerning result could be related to the longer egg development and incubation periods of bearded vultures compared with poultry, and/or due to contrasting environmental conditions during incubation between bearded vultures and poultry. Thus, embryo infection with IBD may occurs via the female or during incubation as a consequence of egg contact between the egg and poultry remains in the nests of bearded vultures, which requires more research.", "Thus, embryo infection with IBD may occurs via the female or during incubation as a consequence of egg contact between the egg and poultry remains in the nests of bearded vultures, which requires more research. Despite their potential effects on population dynamics and conservation through a reduction of productivity and changes in mating behaviour [13, 14] , habitat saturation processes were apparently not directly related to particular proximate causes of egg and nestling failure in this study or in these sampled individuals. As an alternative non-mutually exclusive explanation, we suggest that the recent decline in productivity could also be linked to the increasing ingestion of veterinary drugs and acquisition of pathogens from medicated stabled livestock carcasses due to decreasing availability of unstabled livestock carcasses -the traditional primary food of bearded vultures [16] since the BSE crisis [21] , accompanied by a possible increasing use of antibiotics in stabled livestock operations.", "As an alternative non-mutually exclusive explanation, we suggest that the recent decline in productivity could also be linked to the increasing ingestion of veterinary drugs and acquisition of pathogens from medicated stabled livestock carcasses due to decreasing availability of unstabled livestock carcasses -the traditional primary food of bearded vultures [16] since the BSE crisis [21] , accompanied by a possible increasing use of antibiotics in stabled livestock operations. In this sense, it is remarkable that bearded vultures primarily feed upon livestock bones, which are one of the major target tissues of fluoroquinolones in medicated animals [56] , therefore, rendering this species especially sensitive to the consequences of an increase in the consumption of stabled intensively medicated livestock. The presence of veterinary drugs in eggs implies their previous presence at least in breeding females [12] , but also probably in breeding males and non-breeders frequently using artificial feeding sites and livestock carcass dumps [17] , where veterinary drugs may be ingested from medicated livestock carcasses [10, 21] .", "The presence of veterinary drugs in eggs implies their previous presence at least in breeding females [12] , but also probably in breeding males and non-breeders frequently using artificial feeding sites and livestock carcass dumps [17] , where veterinary drugs may be ingested from medicated livestock carcasses [10, 21] . Therefore, further research is required to determine the impact of veterinary drugs and livestock disease on fitness of full-grown individuals, including the potentially subtle, sublethal or indirect effects of these factors on population dynamics. The link between veterinary drugs and livestock disease should be further investigated in scavenger species, because both threats may concur in food and because the immunodepressive effects and other physiological alterations caused by drugs may facilitate the acquisition and proliferation of pathogens [6, 11, 21] .", "The link between veterinary drugs and livestock disease should be further investigated in scavenger species, because both threats may concur in food and because the immunodepressive effects and other physiological alterations caused by drugs may facilitate the acquisition and proliferation of pathogens [6, 11, 21] . Given that both threats acting together may greatly contribute to breeding failure decreasing productivity, their potential as stochastic factors with potentially devastating effects increasing the risk of extinction should be not overlooked in current conservation programs of bearded vultures and other scavenger species, especially regarding dangerous veterinary drugs and highly pathogenic viruses frequently infecting poultry. In addition, restricted geographic distribution and low genetic variability [57] common to many threatened species may favour pathogen transmission and reduce the ability of a naïve immune system to fight against novel pathogens [3, 28, 58] , making them especially vulnerable to the potential cross-species transmission of highly virulent virus strains able to cause important outbreaks, as reported in poultry [59] [60] [61] .", "In addition, restricted geographic distribution and low genetic variability [57] common to many threatened species may favour pathogen transmission and reduce the ability of a naïve immune system to fight against novel pathogens [3, 28, 58] , making them especially vulnerable to the potential cross-species transmission of highly virulent virus strains able to cause important outbreaks, as reported in poultry [59] [60] [61] . The association of pollution and disease may further increase extinction risk if it interacts with the effects of habitat saturation processes [13, 14, 17] . These processes may facilitate conspecific contact and interactions also likely to increase intra-and interspecific pathogen transmission rates in breeding and feeding areas, especially of highly contagious poultry diseases [22] .", "These processes may facilitate conspecific contact and interactions also likely to increase intra-and interspecific pathogen transmission rates in breeding and feeding areas, especially of highly contagious poultry diseases [22] . This could be further enhanced by the artificially high numbers of bearded vultures and other scavengers attracted to feeding points and carcass refuse dumps, both as a result of management and due to the scarcity of unstabled livestock carcasses since the BSE crisis [17, 21] . Whatever the potential contribution of underlying ultimate mechanisms reducing productivity, our findings highlight the need to determine the proximate causes of breeding failure and mortality in wildlife populations in order to understand the processes regulating demography from an ecological framework perspective.", "Whatever the potential contribution of underlying ultimate mechanisms reducing productivity, our findings highlight the need to determine the proximate causes of breeding failure and mortality in wildlife populations in order to understand the processes regulating demography from an ecological framework perspective. Table S1\n\nFound at: .1371/journal.pone.0014163.s001" ]

[ "There is increasing concern about the impact of veterinary drugs and livestock pathogens as factors damaging wildlife health, especially of threatened avian scavengers feeding upon medicated livestock carcasses. We conducted a comprehensive study of failed eggs and dead nestlings in bearded vultures (Gypaetus barbatus) to attempt to elucidate the proximate causes of breeding failure behind the recent decline in productivity in the Spanish Pyrenees. We found high concentrations of multiple veterinary drugs, primarily fluoroquinolones, in most failed eggs and nestlings, associated with multiple internal organ damage and livestock pathogens causing disease, especially septicaemia by swine pathogens and infectious bursal disease.", "We found high concentrations of multiple veterinary drugs, primarily fluoroquinolones, in most failed eggs and nestlings, associated with multiple internal organ damage and livestock pathogens causing disease, especially septicaemia by swine pathogens and infectious bursal disease. The combined impact of drugs and disease as stochastic factors may result in potentially devastating effects exacerbating an already high risk of extinction and should be considered in current conservation programs for bearded vultures and other scavenger species, especially in regards to dangerous veterinary drugs and highly pathogenic poultry viruses. Text: Environmental pollutants are increasingly documented as a driver of wildlife endangerment due to their roles in organ damage, hormonal disruption and alteration of the immune system [1, 2] .", "Text: Environmental pollutants are increasingly documented as a driver of wildlife endangerment due to their roles in organ damage, hormonal disruption and alteration of the immune system [1, 2] . Disease may also facilitate endangerment and extinction at global and local scales, especially when pathogens interact with other drivers such as pollutants [3] . There is increasing concern about the impact of veterinary drugs and livestock pathogens as factors damaging wildlife health [4] [5] [6] , and even causing declines approaching extinction [7] .", "There is increasing concern about the impact of veterinary drugs and livestock pathogens as factors damaging wildlife health [4] [5] [6] , and even causing declines approaching extinction [7] . These threats may be especially detrimental to wildlife as they increasingly concur and interact as a consequence of the elimination of livestock residues containing veterinary pharmaceuticals and resistant pathogens due to growing intensive livestock operations worldwide [6, 8, 9] . In particular, the ingestion of antimicrobials, primarily fluoroquinolones, has been recently related to immunodepression-mediated acquisition of opportunistic pathogens and disease, as well as to organ damage in nestling vultures [6, 10, 11] .", "In particular, the ingestion of antimicrobials, primarily fluoroquinolones, has been recently related to immunodepression-mediated acquisition of opportunistic pathogens and disease, as well as to organ damage in nestling vultures [6, 10, 11] . Fluoroquinolone residues have also been found in avian scavenger eggs and are associated with severe alterations in the development of embryo cartilage and bones that could preclude embryo movement and subsequently normal development, pre-hatch position and successful hatching [12] . Therefore, antimicrobials and other drugs may negatively affect embryo and nestling health with potentially devastating consequences on breeding success and conservation of vultures and other threatened avian scavengers.", "Therefore, antimicrobials and other drugs may negatively affect embryo and nestling health with potentially devastating consequences on breeding success and conservation of vultures and other threatened avian scavengers. The bearded vulture (Gypaetus barbatus) is one of the most endangered birds in Europe, with a main stronghold in the Pyrenees. Increasing declines in productivity (average number of fledglings raised per territorial pair) have recently been reported in the Spanish Pyrenees associated with habitat saturation processes [13, 14] .", "Increasing declines in productivity (average number of fledglings raised per territorial pair) have recently been reported in the Spanish Pyrenees associated with habitat saturation processes [13, 14] . Given that bearded vultures may raise only one fledgling per breeding attempt, this productivity decline should be linked to increasing breeding failure when the proportion of territorial pairs that are breeding does not greatly vary with time [15] . The proximate mechanisms by which density can affect productivity have been investigated, including habitat heterogeneity, with progressively poorer territories being used, territory shrinkage and interference with breeders and floaters [13] .", "The proximate mechanisms by which density can affect productivity have been investigated, including habitat heterogeneity, with progressively poorer territories being used, territory shrinkage and interference with breeders and floaters [13] . However, the proximate causes of breeding failure are poorly known despite the long-term interests in the conservation of this species [16] . To evaluate these causes, the examination of failed eggs and dead nestlings is imperative, including the study of the presence and impact of injury, developmental problems, poor nutritional condition, pollutants, organ damage, pathogens causing disease, etc.", "To evaluate these causes, the examination of failed eggs and dead nestlings is imperative, including the study of the presence and impact of injury, developmental problems, poor nutritional condition, pollutants, organ damage, pathogens causing disease, etc. in order to determine the most likely cause of breeding failure. Here, we conducted a comprehensive study of failed eggs and dead nestling bearded vultures collected during recent years in the Pyrenees.", "Here, we conducted a comprehensive study of failed eggs and dead nestling bearded vultures collected during recent years in the Pyrenees. Both the productivity and survival rates of adults and young birds have reached the lowest values since the bovine spongiform encephalopathy (BSE) crisis [13, 14, 17] . This temporal decline could be related to illegal poisoning [17] and recent changes in the abundance, distribution and quality of carrion available to avian scavengers as a consequence of EU regulations derived from the BSE crisis [6, [18] [19] [20] .", "This temporal decline could be related to illegal poisoning [17] and recent changes in the abundance, distribution and quality of carrion available to avian scavengers as a consequence of EU regulations derived from the BSE crisis [6, [18] [19] [20] . In particular, the BSE crisis caused the lack or scarcity of unstabled livestock available to scavengers and their subsequent increase in the consumption of carrion from stabled livestock, which is intensively medicated [21] . Therefore, we specifically focused on determining whether breeding failure in bearded vultures is related to the ingestion of veterinary drugs from stabled livestock carrion, as documented in other avian scavenger species [12] .", "Therefore, we specifically focused on determining whether breeding failure in bearded vultures is related to the ingestion of veterinary drugs from stabled livestock carrion, as documented in other avian scavenger species [12] . We also assessed the potential effects of veterinary drugs on embryo damage and immunodepression increasing the probability of acquisition and proliferation of pathogens causing fatal disease [6, [10] [11] [12] 21] . Because veterinary drugs should be exclusively acquired from the ingestion of carrion from livestock medicated to combat disease, we predict that their presence should be associated with that of pathogens acquired from the same livestock, especially poultry pathogens more likely transmitted between avian species [22] .", "Because veterinary drugs should be exclusively acquired from the ingestion of carrion from livestock medicated to combat disease, we predict that their presence should be associated with that of pathogens acquired from the same livestock, especially poultry pathogens more likely transmitted between avian species [22] . Alternatively, if the temporal decline in productivity was primarily associated with breeding failure due to the effects of habitat saturation processes [13, 17] , we should expect egg and nestling mortality to be directly related to developmental and nutritional problems indicating progressively lower quality territories (e.g. embryo emaciation, nestling starvation) and interference by both conspecifics and heterospecifics (e.g.", "embryo emaciation, nestling starvation) and interference by both conspecifics and heterospecifics (e.g. incubation failure, injury due to predation attempts or disturbance). Failed eggs (n = 5) and dead nestlings (n = 4) were collected from bearded vulture nests located in the Spanish Pyrenees between 2005 and 2008.", "Failed eggs (n = 5) and dead nestlings (n = 4) were collected from bearded vulture nests located in the Spanish Pyrenees between 2005 and 2008. The study of this material did not require of the approval of an ethics committee because it was collected after breeding failure (egg or nestling death) was confirmed in the field. Three of the specimens (two nestlings and one egg) were collected in 2005, 2007 and 2008 from a particular territory.", "Three of the specimens (two nestlings and one egg) were collected in 2005, 2007 and 2008 from a particular territory. Eggs and nestlings were collected after breeding failure and frozen. Necropsies were performed on all specimens according to standard protocols [12] . The age of embryos and nestlings were estimated according to size and development.", "The age of embryos and nestlings were estimated according to size and development. Samples of liver, kidney, spleen, large and small intestines, lungs, brain, lymphoid organs (thymus, bursa of Fabricius, Peyer's patches) and knee joints were fixed in 10% buffered formalin, sectioned at 4 mm and stained for histopathological analysis [10, 12] . Liver (dead nestlings and failed embryos) and yolk (failed embryos) were used for the determination of the presence of veterinary drugs, including fluoroquinolones (enrofloxacin and ciprofloxacin), other antimicrobials (amoxicillin and oxytetracycline), non-steroideal anti-inflamatories (NSAIDs) such as diclofenac, flunixin meglumine, ketoprofen, ibuprofen, meloxicam, sodium salicylate, acetaminophen, and antiparasitics (metronidazole, diclazuril, fenbendazole, ivermectin) as described previously [12] .", "Liver (dead nestlings and failed embryos) and yolk (failed embryos) were used for the determination of the presence of veterinary drugs, including fluoroquinolones (enrofloxacin and ciprofloxacin), other antimicrobials (amoxicillin and oxytetracycline), non-steroideal anti-inflamatories (NSAIDs) such as diclofenac, flunixin meglumine, ketoprofen, ibuprofen, meloxicam, sodium salicylate, acetaminophen, and antiparasitics (metronidazole, diclazuril, fenbendazole, ivermectin) as described previously [12] . The limits of quantification, percentage recoveries, and interand intra-assay reproducibility were adequate [10, 12] . Other contaminants potentially affecting eggs and embryos were determined in liver, including heavy metals (Cd, Zn, Pb and Hg), following Blanco et al.", "Other contaminants potentially affecting eggs and embryos were determined in liver, including heavy metals (Cd, Zn, Pb and Hg), following Blanco et al. [23] , dithiocarbamate thiram, disulfuram, polybrominated diphenyl ethers, organochlorines and brominated flame retardants, following Lemus et al. [12] and carbamate and organophosphate pesticides (carbofuran, aldicarb and fenthion) following Elliot et al.", "[12] and carbamate and organophosphate pesticides (carbofuran, aldicarb and fenthion) following Elliot et al. [24] . We measured brain cholinesterase activity to assess early exposure to anticholinesterase pesticides [25] .", "We measured brain cholinesterase activity to assess early exposure to anticholinesterase pesticides [25] . Potential contamination was assessed by comparison with levels from apparently normal wild birds of other species [26] in the absence of basal levels for bearded vultures. Determination of bacterial and fungal pathogens were conducted by sampling oropharynx, lung, liver, kidney, spleen, and intestine with sterile swabs and cultured using standard microbiology protocols [10, 12, 27, 28] .", "Determination of bacterial and fungal pathogens were conducted by sampling oropharynx, lung, liver, kidney, spleen, and intestine with sterile swabs and cultured using standard microbiology protocols [10, 12, 27, 28] . Salmonella serotypes and phage types were determined in the Spanish Reference Laboratory (Laboratorio Central Veterinario, Algete, Madrid). For confirmation of the identification of the alpha hemolytic Streptococcus pneumoniae we used a specific identification test (Accuprobe, Salem, MA) based on the detection of specific ribosomal RNA sequences.", "For confirmation of the identification of the alpha hemolytic Streptococcus pneumoniae we used a specific identification test (Accuprobe, Salem, MA) based on the detection of specific ribosomal RNA sequences. Samples of lesions found in internal organs and tissues during necropsies were taken with sterile swabs and cultured using the same standard microbiology protocols. In addition, we determined the presence of selected avian pathogens, including bacterial, viral, fungal, and protozoan pathogens by means of PCR-based methods (see Table S1 for details).", "In addition, we determined the presence of selected avian pathogens, including bacterial, viral, fungal, and protozoan pathogens by means of PCR-based methods (see Table S1 for details). The presence of Chlamyophila psittaci and Mycoplasma sp. in blood was determined as described previously [29, 30] .", "in blood was determined as described previously [29, 30] . The presence of poxvirus, the paramyxovirus causing Newcastle disease, the serotypes H5, H7 and H9 of avian influenza, falcon adenovirus, circovirus, herpesvirus, polyomavirus, reovirus and West Nile virus were determined following the PCR-based methods available in the literature [31] [32] [33] [34] [35] [36] [37] [38] [39] . We also searched for helminths and protozoans in the gastrointestinal tract by macroscopic and microscopic observations using standard protocols [40] .", "We also searched for helminths and protozoans in the gastrointestinal tract by macroscopic and microscopic observations using standard protocols [40] . Specific immunocytochemical procedures were used for detection of mielodepressive virus, including the alphaherpesvirus causing Marek disease [41] in kidney and bursa of Fabricius, the gyrovirus causing infectious chicken anaemia [42] in thymus and bone marrow, the birnavirus causing infectious bursal disease (IBD, [43] ) in bursa of Fabricius, and the coronavirus causing chicken infectious bronchitis in kidney [44] . In addition, we conducted a specific immunocytochemical procedure for West Nile virus antigen detection [45] in brain, spinal medulla, thymus and thyroid.", "In addition, we conducted a specific immunocytochemical procedure for West Nile virus antigen detection [45] in brain, spinal medulla, thymus and thyroid. All immunohistochemistry analyses were conducted at the Department of Veterinary Anatomy, Veterinary Faculty, Universidad Complutense de Madrid, Spain and at the Pathology Department of the Veterinary Faculty, University of Utrecht, The Netherlands. The presence of these viruses was also determined by PCR-based methods [43, [46] [47] [48] .", "The presence of these viruses was also determined by PCR-based methods [43, [46] [47] [48] . All dead nestlings and three of five unhatched embryos showed two to six different veterinary drugs in liver (nestlings) and egg yolk (embryos). In addition, the two embryos with fluoroquinolones in the yolk also had them in the liver (Table 1) .", "In addition, the two embryos with fluoroquinolones in the yolk also had them in the liver (Table 1) . Fluoroquinolones were the most prevalent drugs and showed the highest concentrations (Table 1) . Other drugs such as NSAIDs and antiparasitics were found in most nestlings at variable concentrations, but in no eggs (Table 1) .", "Other drugs such as NSAIDs and antiparasitics were found in most nestlings at variable concentrations, but in no eggs (Table 1) . Other toxic compounds were detected in lower prevalence and concentrations (see Table 1 for those more relevant values; all insecticides were found at concentrations ,0.001 ppb), which was further supported by basal levels of brain cholinesterase (Table 1) . Dead embryos and nestlings showed a moderate to good nutritional state.", "Dead embryos and nestlings showed a moderate to good nutritional state. Major histopathological lesions were primarily located in the kidney, including glomerulonephritis and/or glomerulonephrosis present in all individuals with fluoroquinolones, but not in those without drugs (Table 1 ). All individuals with fluoroquinolones also showed joint lesions, including arthritis and/ or arthrosis of the long bone articulations, as well as massive osseous stroma of the spongeous bones.", "All individuals with fluoroquinolones also showed joint lesions, including arthritis and/ or arthrosis of the long bone articulations, as well as massive osseous stroma of the spongeous bones. The fungi Candida albicans was isolated from the oral cavity of five individuals. All individuals showed non-specific mixedbacterial flora. Enterotoxigenic Escherichia coli and Salmonella spp.", "All individuals showed non-specific mixedbacterial flora. Enterotoxigenic Escherichia coli and Salmonella spp. were isolated in four cases ( Table 1 (cont.) *Samples from the same territory in different years. 1 Veterinary drugs.", "*Samples from the same territory in different years. 1 Veterinary drugs. EN: enrofloxacin (mg/g), CI: ciprofloxacin (mg/g), OX: oxytetracyclin (mg/g), FL: flunixin meglumine (mg/g), AS: sodium salicylate (ng/g), IV: Ivermectin (mg/g). 2 Other toxicants.", "2 Other toxicants. OR: organochlorines (ng/g), Pb: lead (ng/g). 12. z29 (three cases, see Table 1 ).", "12. z29 (three cases, see Table 1 ). One individual showed infection by Salmonella enterica enteritidis (see above) and enterotoxigenic Escherichia coli O86 in all examined organs (septicaemia) except brain, which rejected the possibility of post-mortem contamination. Pasteurella multocida was isolated in a single individual that also showed enterotoxigenic Escherichia coli O86 (Table 1) ; all of these individuals contained fluoroquinolones.", "Pasteurella multocida was isolated in a single individual that also showed enterotoxigenic Escherichia coli O86 (Table 1) ; all of these individuals contained fluoroquinolones. One of the failed embryos without veterinary drugs showed suppurative myocarditis, multiple microabscesses in head muscles, suppurative leptomeningitis, as well as lower jaw gangrenous inflammation with loss of the osseous stroma due to a mixed infection with Streptococcus suis and Streptococcus pneumoniae in brain, meninges and neck muscles; this embryo also showed infection by chicken infectious bronchitis ( Table 1 ). Both immunocytochemistry for the detection of poultry viruses and PCR pathogen survey were positive to IBDV in six individuals with fluoroquinolones (Table 1) .", "Both immunocytochemistry for the detection of poultry viruses and PCR pathogen survey were positive to IBDV in six individuals with fluoroquinolones (Table 1) . Immunocytochemical procedures failed to detect West Nile virus antigens in individuals in which PCR for this virus had been positive. Parasitology was negative for all helminths, helminth eggs and protozoans.", "Parasitology was negative for all helminths, helminth eggs and protozoans. We found multiple veterinary drugs, primarily fluoroquinolones, in most failed eggs and dead nestling bearded vultures from the Pyrenees. They also showed multiple internal organ damage and pathogens potentially acquired from medicated livestock carrion, especially viruses often infecting poultry.", "They also showed multiple internal organ damage and pathogens potentially acquired from medicated livestock carrion, especially viruses often infecting poultry. Recorded drug concentrations were among the highest reported in avian scavengers [6, [10] [11] [12] 21] . NSAIDs and antiparasitics were found in lower prevalence than fluoroquinolones, but at higher concentrations than those found in other avian scavengers, especially for flunixin meglumine and sodium salicylate [6, 12, 21] .", "NSAIDs and antiparasitics were found in lower prevalence than fluoroquinolones, but at higher concentrations than those found in other avian scavengers, especially for flunixin meglumine and sodium salicylate [6, 12, 21] . On the contrary, we found no sterile eggs, poor nutritional conditions or injury in any failed embryo or nestling. Other pollutants were found in low prevalence and concentrations posing low risk to embryo and nestling health.", "Other pollutants were found in low prevalence and concentrations posing low risk to embryo and nestling health. Fluoroquinolones may cause generalized direct developmental damage precluding embryo hatching, physiological alterations due to their impact on liver and kidney and immunodepression reducing resistance to opportunistic pathogens [6, [10] [11] [12] 21] . These pathogens may be acquired at the same time that drugs used to treat diseased livestock are ingested, as indicated by their high prevalence in embryos and nestlings.", "These pathogens may be acquired at the same time that drugs used to treat diseased livestock are ingested, as indicated by their high prevalence in embryos and nestlings. Therefore, despite the relatively small sample size resulting from low abundance, endangerment and logistic difficulties in reaching nests in this species, the results provide evidence of a combined impact of veterinary drugs and livestock disease as the primary cause of breeding failure in the sampled individuals. The presence of West Nile virus is not likely to be associated with nestling disease or mortality because the lack of lesions in target tissues and viral antigen particles in the immunohistochemistry study.", "The presence of West Nile virus is not likely to be associated with nestling disease or mortality because the lack of lesions in target tissues and viral antigen particles in the immunohistochemistry study. Fatal septicaemia caused by Streptococcus suis, one of the most important swine pathogens worldwide [49] , in combination with septicaemia from Streptococcus pneumoniae and infection by chicken infectious bronchitis virus were found in a single embryo. This concentration of livestock pathogens has not been reported before and, to our knowledge, this is the first report of the three pathogens causing disease in a wild bird.", "This concentration of livestock pathogens has not been reported before and, to our knowledge, this is the first report of the three pathogens causing disease in a wild bird. Other pathogens recorded in embryos and nestlings, including Salmonella serotypes and phages typical of livestock [50] , and enterotoxigenic Escherichia coli O86 causing septicaemia, were potentially transmitted by consumption of carcasses of infected poultry and other livestock [22, 27, 28] . In addition, we found that the IBD virus infected most individuals alone or together with other pathogens also potentially acquired from livestock carrion.", "In addition, we found that the IBD virus infected most individuals alone or together with other pathogens also potentially acquired from livestock carrion. This virus causes a highly contagious immunosuppressive bursal disease in poultry [51] and may be transmitted to wildlife in contact with poultry waste or by ingestion of carcasses [22, 52] . Nestlings are especially susceptible to IBD because of the primary role of bursa of Fabricius in immune function development at this age.", "Nestlings are especially susceptible to IBD because of the primary role of bursa of Fabricius in immune function development at this age. In fact, immunosuppression due to IBD was indicated by the inflammation, necrosis and loss of lymphocytes in the bursa of Fabricius together with the presence of viral antigens recorded by means of immunocytochemical procedures. The potential impact of highly pathogenic and contagious poultry viruses has been previously recognized as a threat to wildlife health due to the increasing contact of wildlife with livestock operations in general, and poultry farms and their residues in particular, in natural areas worldwide [52] [53] [54] [55] .", "The potential impact of highly pathogenic and contagious poultry viruses has been previously recognized as a threat to wildlife health due to the increasing contact of wildlife with livestock operations in general, and poultry farms and their residues in particular, in natural areas worldwide [52] [53] [54] [55] . However, damage from IBD virus on the bursa of Fabricius represents, to our knowledge, the first evidence of clinical disease compatible with death caused by this poultry virus in wildlife. The presence of IBD has been not previously recorded in embryos of wild birds, probably because vertical transmission has been ruled out in poultry and, as consequence, it has probably not been evaluated in other species until now.", "The presence of IBD has been not previously recorded in embryos of wild birds, probably because vertical transmission has been ruled out in poultry and, as consequence, it has probably not been evaluated in other species until now. This striking and concerning result could be related to the longer egg development and incubation periods of bearded vultures compared with poultry, and/or due to contrasting environmental conditions during incubation between bearded vultures and poultry. Thus, embryo infection with IBD may occurs via the female or during incubation as a consequence of egg contact between the egg and poultry remains in the nests of bearded vultures, which requires more research.", "Thus, embryo infection with IBD may occurs via the female or during incubation as a consequence of egg contact between the egg and poultry remains in the nests of bearded vultures, which requires more research. Despite their potential effects on population dynamics and conservation through a reduction of productivity and changes in mating behaviour [13, 14] , habitat saturation processes were apparently not directly related to particular proximate causes of egg and nestling failure in this study or in these sampled individuals. As an alternative non-mutually exclusive explanation, we suggest that the recent decline in productivity could also be linked to the increasing ingestion of veterinary drugs and acquisition of pathogens from medicated stabled livestock carcasses due to decreasing availability of unstabled livestock carcasses -the traditional primary food of bearded vultures [16] since the BSE crisis [21] , accompanied by a possible increasing use of antibiotics in stabled livestock operations.", "As an alternative non-mutually exclusive explanation, we suggest that the recent decline in productivity could also be linked to the increasing ingestion of veterinary drugs and acquisition of pathogens from medicated stabled livestock carcasses due to decreasing availability of unstabled livestock carcasses -the traditional primary food of bearded vultures [16] since the BSE crisis [21] , accompanied by a possible increasing use of antibiotics in stabled livestock operations. In this sense, it is remarkable that bearded vultures primarily feed upon livestock bones, which are one of the major target tissues of fluoroquinolones in medicated animals [56] , therefore, rendering this species especially sensitive to the consequences of an increase in the consumption of stabled intensively medicated livestock. The presence of veterinary drugs in eggs implies their previous presence at least in breeding females [12] , but also probably in breeding males and non-breeders frequently using artificial feeding sites and livestock carcass dumps [17] , where veterinary drugs may be ingested from medicated livestock carcasses [10, 21] .", "The presence of veterinary drugs in eggs implies their previous presence at least in breeding females [12] , but also probably in breeding males and non-breeders frequently using artificial feeding sites and livestock carcass dumps [17] , where veterinary drugs may be ingested from medicated livestock carcasses [10, 21] . Therefore, further research is required to determine the impact of veterinary drugs and livestock disease on fitness of full-grown individuals, including the potentially subtle, sublethal or indirect effects of these factors on population dynamics. The link between veterinary drugs and livestock disease should be further investigated in scavenger species, because both threats may concur in food and because the immunodepressive effects and other physiological alterations caused by drugs may facilitate the acquisition and proliferation of pathogens [6, 11, 21] .", "The link between veterinary drugs and livestock disease should be further investigated in scavenger species, because both threats may concur in food and because the immunodepressive effects and other physiological alterations caused by drugs may facilitate the acquisition and proliferation of pathogens [6, 11, 21] . Given that both threats acting together may greatly contribute to breeding failure decreasing productivity, their potential as stochastic factors with potentially devastating effects increasing the risk of extinction should be not overlooked in current conservation programs of bearded vultures and other scavenger species, especially regarding dangerous veterinary drugs and highly pathogenic viruses frequently infecting poultry. In addition, restricted geographic distribution and low genetic variability [57] common to many threatened species may favour pathogen transmission and reduce the ability of a naïve immune system to fight against novel pathogens [3, 28, 58] , making them especially vulnerable to the potential cross-species transmission of highly virulent virus strains able to cause important outbreaks, as reported in poultry [59] [60] [61] .", "In addition, restricted geographic distribution and low genetic variability [57] common to many threatened species may favour pathogen transmission and reduce the ability of a naïve immune system to fight against novel pathogens [3, 28, 58] , making them especially vulnerable to the potential cross-species transmission of highly virulent virus strains able to cause important outbreaks, as reported in poultry [59] [60] [61] . The association of pollution and disease may further increase extinction risk if it interacts with the effects of habitat saturation processes [13, 14, 17] . These processes may facilitate conspecific contact and interactions also likely to increase intra-and interspecific pathogen transmission rates in breeding and feeding areas, especially of highly contagious poultry diseases [22] .", "These processes may facilitate conspecific contact and interactions also likely to increase intra-and interspecific pathogen transmission rates in breeding and feeding areas, especially of highly contagious poultry diseases [22] . This could be further enhanced by the artificially high numbers of bearded vultures and other scavengers attracted to feeding points and carcass refuse dumps, both as a result of management and due to the scarcity of unstabled livestock carcasses since the BSE crisis [17, 21] . Whatever the potential contribution of underlying ultimate mechanisms reducing productivity, our findings highlight the need to determine the proximate causes of breeding failure and mortality in wildlife populations in order to understand the processes regulating demography from an ecological framework perspective.", "Whatever the potential contribution of underlying ultimate mechanisms reducing productivity, our findings highlight the need to determine the proximate causes of breeding failure and mortality in wildlife populations in order to understand the processes regulating demography from an ecological framework perspective. Table S1\n\nFound at: .1371/journal.pone.0014163.s001" ]

[ "BACKGROUND: Sustained outbreaks of highly pathogenic avian influenza (HPAI) H5N1 in avian species increase the risk of reassortment and adaptation to humans. The ability to contain its spread in chickens would reduce this threat and help maintain the capacity for egg-based vaccine production. While vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses.", "While vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses. METHODOLOGY / PRINCIPAL FINDINGS: The ability of DNA vaccines encoding hemagglutinin (HA) proteins from different HPAI H5N1 serotypes was evaluated for its ability to elicit neutralizing antibodies and to protect against homologous and heterologous HPAI H5N1 strain challenge in mice and chickens after DNA immunization by needle and syringe or with a pressure injection device. These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs.", "These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs. The response was dose-dependent, and breadth was determined by the choice of the influenza virus HA in the vaccine. Monovalent and trivalent HA vaccines were tested first in mice and conferred protection against lethal H5N1 A/Vietnam/1203/2004 challenge 68 weeks after vaccination.", "Monovalent and trivalent HA vaccines were tested first in mice and conferred protection against lethal H5N1 A/Vietnam/1203/2004 challenge 68 weeks after vaccination. In chickens, protection was observed against heterologous strains of HPAI H5N1 after vaccination with a trivalent H5 serotype DNA vaccine with doses as low as 5 µg DNA given twice either by intramuscular needle injection or with a needle-free device. CONCLUSIONS/SIGNIFICANCE: DNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species.", "CONCLUSIONS/SIGNIFICANCE: DNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. In addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates. Text: The highly pathogenic H5N1 influenza virus causes lethal multi-organ disease in poultry, resulting in significant economic losses and a public health concern in many parts of the world.", "Text: The highly pathogenic H5N1 influenza virus causes lethal multi-organ disease in poultry, resulting in significant economic losses and a public health concern in many parts of the world. The greatest threats posed by this virus are its ability to cause mortality in humans, its potential to compromise food supplies, and its possible economic impacts. Viral maintenance in poultry potentiates the risk of human-to-human transmission and the emergence of a pandemic strain through reassortment.", "Viral maintenance in poultry potentiates the risk of human-to-human transmission and the emergence of a pandemic strain through reassortment. An effective, safe poultry vaccine that elicits broadly protective immune responses to evolving flu strains would provide a countermeasure to reduce the likelihood of transmission of this virus from domestic birds to humans and simultaneously would protect commercial poultry operations and subsistence farmers. DNA vaccines have been shown to elicit robust immune responses in various animal species, from mice to nonhuman primates [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] .", "DNA vaccines have been shown to elicit robust immune responses in various animal species, from mice to nonhuman primates [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] . In human trials, these vaccines elicit cellular and humoral immune responses against various infectious agents, including influenza, SARS, SIV and HIV. In addition to their ability to elicit antibody responses, they also stimulate antigenspecific and sustained T cell responses [1] [2] [3] 6, 12, 13] .", "In addition to their ability to elicit antibody responses, they also stimulate antigenspecific and sustained T cell responses [1] [2] [3] 6, 12, 13] . DNA vaccination has been used experimentally against various infectious agents in a variety of mammals, including cattle (against infectious bovine rhinotracheitis/bovine diarrhea virus, leptospirosis and mycobacteriosis) [14, 15] , pigs (against classical swine fever virus and mycoplasmosis) [16] , and horses (against West Nile virus and rabies) [17] . In addition, DNA vaccines have been tested against avian plasmodium infection in penguins [18] and against influenza and infectious bursal disease in chickens [7, 8, 19] , duck hepatitis B virus in ducks [6] , and avian metapneumovirus and Chlamydia psittaci in turkeys [20, 21] (reviewed in ref.", "In addition, DNA vaccines have been tested against avian plasmodium infection in penguins [18] and against influenza and infectious bursal disease in chickens [7, 8, 19] , duck hepatitis B virus in ducks [6] , and avian metapneumovirus and Chlamydia psittaci in turkeys [20, 21] (reviewed in ref. [22] ). While they have been used in chickens to generate antisera to specific influenza viruses and confer protection against the low pathogenicity H5N2 strain [23] , there is only one previous report of a monovalent DNA vaccine effective against H5N1 (and that only against a matched H5N1 isolate) [24] ; no protection with multivalent DNA vaccines against heterologous strains has been reported.", "While they have been used in chickens to generate antisera to specific influenza viruses and confer protection against the low pathogenicity H5N2 strain [23] , there is only one previous report of a monovalent DNA vaccine effective against H5N1 (and that only against a matched H5N1 isolate) [24] ; no protection with multivalent DNA vaccines against heterologous strains has been reported. Development and characterization of a DNA vaccine modality for use in poultry offers a potential countermeasure against HPAI H5N1 avian influenza outbreaks. The virus can infect humans, typically from animal sources, including commercial and wild avian species, livestock, and possibly other non-domesticated animal species [25] [26] [27] .", "The virus can infect humans, typically from animal sources, including commercial and wild avian species, livestock, and possibly other non-domesticated animal species [25] [26] [27] . While there is marked diversity in the host range of type A influenza viruses, many experts have speculated that a pandemic strain of type A influenza could evolve in avian species or avian influenza viruses could contribute virulent genes to a pandemic strain through reassortment [28, 29] . Thus, there is reason to consider vaccination of poultry that would stimulate potent and broad protective immune responses [7, 30, 31] .", "Thus, there is reason to consider vaccination of poultry that would stimulate potent and broad protective immune responses [7, 30, 31] . In undertaking such efforts, it is important that there be a differentiation of infected from vaccinated animals [32] so that animals can be protected and permit monitoring of new infections using proven and sensitive methodologies. In this study, we used an automated high capacity needle-free injection device, Agro-JetH (Medical International Technology, Inc., Denver, CO) to explore the feasibility of DNA vaccination of poultry.", "In this study, we used an automated high capacity needle-free injection device, Agro-JetH (Medical International Technology, Inc., Denver, CO) to explore the feasibility of DNA vaccination of poultry. After optimization of injection conditions, alternative multivalent DNA vaccine regimens were analyzed and compared for magnitude and breadth of neutralizing antibodies, as well as protective efficacy after challenge in mouse and chicken models of HPAI H5N1 infection. The findings suggest that it is possible to develop a multivalent DNA vaccine for poultry that can protect against multiple HPAI H5N1 strains and that could keep pace with the continued evolution of avian influenza viruses.", "The findings suggest that it is possible to develop a multivalent DNA vaccine for poultry that can protect against multiple HPAI H5N1 strains and that could keep pace with the continued evolution of avian influenza viruses. Immunogenicity and neutralizing antibody specificity of alternative HA DNA vaccines in mice To evaluate the efficacy of multivalent DNA vaccines, initial studies were performed in mice. Expression vectors encoding HAs from ten phylogenetically diverse strains of influenza viruses [33] were generated by synthesis of cDNAs (see Materials and Methods) in plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo [34, 35, 36] .", "Expression vectors encoding HAs from ten phylogenetically diverse strains of influenza viruses [33] were generated by synthesis of cDNAs (see Materials and Methods) in plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo [34, 35, 36] . Animals were immunized with each expression vector intramuscularly (IM) at three week intervals, and the antisera were evaluated on day 14 after the third immunization for their ability to neutralize HPAI H5N1 pseudotyped lentiviral vectors as previously described [35, 36] . We have previously shown that lentiviral assay inhibition (LAI) yields similar results to microneutralization and HAI analyses with higher sensitivity in mice [35, 36] \n\nTo determine whether immunization with multiple HAs simultaneously could expand the breadth of the neutralizing antibody response without significant loss of magnitude, a combination of 10 HA DNA vaccine immunogens was administered IM at proportionally lower concentration (1.5 mg per immunogen) into groups of 10 mice (see Materials and Methods).", "We have previously shown that lentiviral assay inhibition (LAI) yields similar results to microneutralization and HAI analyses with higher sensitivity in mice [35, 36] \n\nTo determine whether immunization with multiple HAs simultaneously could expand the breadth of the neutralizing antibody response without significant loss of magnitude, a combination of 10 HA DNA vaccine immunogens was administered IM at proportionally lower concentration (1.5 mg per immunogen) into groups of 10 mice (see Materials and Methods). Remarkably, despite a log lower DNA concentration of each component, significant neutralizing antibody titers were generated to each of the 10 immunogens, with .80% neutralization against 6 out of 12 H5 HA pseudoviruses at dilutions of up to 1:400 ( Fig. 2A) .", "2A) . To evaluate whether similar breadth of immunity could be generated with fewer immunogens, two different combinations of 5 immunogens were selected, based on the phylogenetic diversity of HA among the avian influenza viruses [33] and the crossreactivity of the neutralizing antibody responses of select individual immunogens (Fig. 1 ).", "1 ). As expected, there were substantial differences in the breadth of neutralization between these two sets of 5 immunogen multivalent vaccines (Fig. 2 , B vs. C).", "2 , B vs. C). In one set, while neutralization of homologous strains was comparable to the monovalent and the 10 immunogen multivalent immune response, fewer cross-reactive antibodies were detected, directed most prominently against A/Iraq Protection of DNA-vaccinated mice against challenge with heterologous H5N1 A/Vietnam/1203/2004 influenza virus Mice immunized as described above were challenged with a heterologous H5N1 virus 68 weeks after the final DNA vaccination. Animals were then challenged with 10 LD 50 of the highly pathogenic A/Vietnam/1203/2004 virus intranasally, and morbidity and mortality were monitored for 21 days after the viral challenge.", "Animals were then challenged with 10 LD 50 of the highly pathogenic A/Vietnam/1203/2004 virus intranasally, and morbidity and mortality were monitored for 21 days after the viral challenge. The control animals, injected with the plasmid expression vector with no insert, died within 10 days of infection. Complete survival was observed in the groups immunized with the 10 component and set 2 of the 5 component multivalent DNA vaccines (Fig.", "Complete survival was observed in the groups immunized with the 10 component and set 2 of the 5 component multivalent DNA vaccines (Fig. 3) . Immunization with HA derived from the A/ Indonesia/05/2005 strain or set 1 of the 5 component multivalent DNA vaccine showed a survival rate approaching 90%.", "Immunization with HA derived from the A/ Indonesia/05/2005 strain or set 1 of the 5 component multivalent DNA vaccine showed a survival rate approaching 90%. In contrast, animals injected with HA plasmid DNA derived from A/ Anhui/1/2005, which has diverged more from A/Vietnam/ 1203/2004, showed a lower percent survival (70%) after lethal viral challenge. Survival differences between groups were assessed using a log-rank test and the Gehan-Wilcoxon test on the survival curves for pairs of groups.", "Survival differences between groups were assessed using a log-rank test and the Gehan-Wilcoxon test on the survival curves for pairs of groups. A test was deemed significant if the pvalue was ,0.01. Mice injected IM with different HAs, A/ Indonesia/5/05, A/Anhui/1/05, 10HA, 5 HA (Set 1), or 5 HA (Set 2) showed a significant difference compared to control (all p values,0.001).", "Mice injected IM with different HAs, A/ Indonesia/5/05, A/Anhui/1/05, 10HA, 5 HA (Set 1), or 5 HA (Set 2) showed a significant difference compared to control (all p values,0.001). Among the HA-immunized groups, there was no significant difference between any two groups (p.0.08 for all comparisons). For example, no significant difference was observed between the A/Anhui/1/05 group, which had the least survival among the HA immunized groups (7 out of 10), and other HA groups: A/Indonesia/5/05 (p = 0.377), 10 HA (p = 0.082), 5 HA (Set 1) (p = 0.101), or 5 HA (Set 2) (p = .411).", "For example, no significant difference was observed between the A/Anhui/1/05 group, which had the least survival among the HA immunized groups (7 out of 10), and other HA groups: A/Indonesia/5/05 (p = 0.377), 10 HA (p = 0.082), 5 HA (Set 1) (p = 0.101), or 5 HA (Set 2) (p = .411). Therefore, we cannot exclude the possibility that the 3 deaths in the A/Anhui/1/05 group may have been due to random chance. Since it is desirable to confer protective immunity in poultry and HA DNA vaccination was effective in mice, we next examined the breadth and potency of single or multiple HA plasmid immunization in chickens.", "Since it is desirable to confer protective immunity in poultry and HA DNA vaccination was effective in mice, we next examined the breadth and potency of single or multiple HA plasmid immunization in chickens. The ability of chickens to generate specific antibodies was assessed with three strains that showed broad cross protection in mouse studies (A/Vietnam/1203/2004, A/Anhui/ 1/2005 and A/Indonesia/05/2005), administered individually or in combination, by different injection methods. In addition to needle injection, a needle-free repetitive injection device, Agro-JetH (Medical International Technology, Inc., Denver, CO), was analyzed.", "In addition to needle injection, a needle-free repetitive injection device, Agro-JetH (Medical International Technology, Inc., Denver, CO), was analyzed. This device disperses the 0.1 to 5 ml injection doses into the dermal, subcutaneous, or intramuscular tissue depending upon the pressure adjustments, powered by a CO 2 gas pressure plunger [39] . The injection conditions were determined by histologic analysis of tissues that received injections of India ink; a pressure of 48 psi was chosen since it enabled consistent delivery into intradermal and subcutaneous tissues (Fig.", "The injection conditions were determined by histologic analysis of tissues that received injections of India ink; a pressure of 48 psi was chosen since it enabled consistent delivery into intradermal and subcutaneous tissues (Fig. S1 ). Immunization of chickens with the control plasmid (CMV/R) without an HA gene insert elicited minimal neutralizing antibody titers compared to HA-immunized animals 1 week after 3 DNA immunizations.", "Immunization of chickens with the control plasmid (CMV/R) without an HA gene insert elicited minimal neutralizing antibody titers compared to HA-immunized animals 1 week after 3 DNA immunizations. Nearly all chickens immunized with either monovalent or multivalent HA DNA vaccines generated significant neutralization titers ( Fig. 4 and Table S1 ).", "4 and Table S1 ). In general, there was a progressive increase in the amount of neutralization after each successive DNA vaccination (data not shown) with maximal response at 1 week after the 3 rd DNA immunization, with highest and most consistent levels in the trivalent vaccine group delivered with the Agro-JetH device. Neutralization of the Indonesia HA strain was the most robust, with neutralization nearing 100% at titers greater than 1:3200.", "Neutralization of the Indonesia HA strain was the most robust, with neutralization nearing 100% at titers greater than 1:3200. Both the monovalent and multivalent vaccines elicited robust homologous ( vaccine (Fig. 4 ).", "4 ). Even though one chicken (238) in the multivalent vaccine group produced almost the same degree of neutralization at each time point and was protected, it did not produce a high neutralizing antibody titer for reasons that were uncertain but possibly related to a non-specific inhibitor in the sera. To determine whether chickens immunized with single or multiple DNA vaccines were protected from a lethal challenge of a heterologous HPAI H5N1 virus, vaccinated chickens were In panels B and C, mice (n = 10) were immunized with 15 mg of plasmid (3 mg each) three times at 3 week intervals.", "To determine whether chickens immunized with single or multiple DNA vaccines were protected from a lethal challenge of a heterologous HPAI H5N1 virus, vaccinated chickens were In panels B and C, mice (n = 10) were immunized with 15 mg of plasmid (3 mg each) three times at 3 week intervals. Serum pools from the immunized animals were collected 14 days after the third immunization. The antisera were tested against the 12 indicated pseudotyped lentiviral vectors at varying dilutions.", "The antisera were tested against the 12 indicated pseudotyped lentiviral vectors at varying dilutions. Error bars at each point indicate the standard deviation; each sample was evaluated in triplicate. In general, the immunized serum neutralized all tested pseudotyped lentiviruses at low dilutions while differences were often observed at high dilution.", "In general, the immunized serum neutralized all tested pseudotyped lentiviruses at low dilutions while differences were often observed at high dilution. .1371/journal.pone.0002432.g002 inoculated with 20 LD 50 of highly pathogenic A/Vietnam/1203/ 2004 heterologous virus intranasally using standard methods [25, 40] and monitored for morbidity, mortality, viral shedding and serum antibodies. While all the control animals died within 2 days of infection, 100% survival was noted in the rest of the chickens (Fig.", "While all the control animals died within 2 days of infection, 100% survival was noted in the rest of the chickens (Fig. 5A ). The animals that were healthy, showing no signs of clinical disease or malaise, were euthanized on day 14.", "The animals that were healthy, showing no signs of clinical disease or malaise, were euthanized on day 14. There was no evidence for viral shedding monitored via tracheal and cloacal swabs of infected chickens 2-14 days after challenge as determined by embryonal inoculation (data not shown: egg infectious dose 50 (EID 50 ) limit of detection ,100 virus particles). To compare the relative efficacy of DNA vaccines delivered IM by needle and syringe versus the needle-free Agro-JetH device injection, a dose-response study was performed with amounts of DNA vaccine ranging from 500 to 0.5 mg with two inoculations.", "To compare the relative efficacy of DNA vaccines delivered IM by needle and syringe versus the needle-free Agro-JetH device injection, a dose-response study was performed with amounts of DNA vaccine ranging from 500 to 0.5 mg with two inoculations. In these experiments, the HA derived from A/chicken/Nigeria/641/ 2006 was substituted for A/Vietnam/1203/2004 since it represented a more contemporary isolate. The observed rate of protection was higher among the animals receiving 5 mg by Agro-Jet (8/8) than by IM injection (6/8) (Fig.", "The observed rate of protection was higher among the animals receiving 5 mg by Agro-Jet (8/8) than by IM injection (6/8) (Fig. 5, B vs. C). Both modes provided complete protection for all animals at doses higher than this, and 25% protection for the animals receiving 0.5 mg doses (Fig.", "Both modes provided complete protection for all animals at doses higher than this, and 25% protection for the animals receiving 0.5 mg doses (Fig. 5B, C) . Survival differences between consecutive doses were assessed using a log-rank test on the survival curves for pairs of groups.", "Survival differences between consecutive doses were assessed using a log-rank test on the survival curves for pairs of groups. A test was deemed significant if the p-value was ,.01, and marginally significant if the p-value was ,.05 but ..01. Chickens injected IM showed a marginally significant difference between 0.5 and 5 mg (p = .047).", "Chickens injected IM showed a marginally significant difference between 0.5 and 5 mg (p = .047). In the same group there was a significant difference between control and 5, 50 and 500 mg (p,.001 for all comparisons) and the difference between control and 0.5 mg was marginally significant (p = .016). Chickens that were injected using Agro-JetH showed a significant difference between 0.5 and 5 mg (p = .004) and between control and 5, 50, and 500 mg (p,.001 for all comparisons).", "Chickens that were injected using Agro-JetH showed a significant difference between 0.5 and 5 mg (p = .004) and between control and 5, 50, and 500 mg (p,.001 for all comparisons). There were no differences between control and 0.5 mg or between 5, 50, and 500 mg. Lastly, the survival differences between Agro-JetH and IM for each dose group were not significant. The neutralizing antibody response to homologous and heterologous HAs corresponded with protection and correlated with dose, with higher titers elicited by injection with Agro-JetH compared to needle (Table S2) .", "The neutralizing antibody response to homologous and heterologous HAs corresponded with protection and correlated with dose, with higher titers elicited by injection with Agro-JetH compared to needle (Table S2) . We assessed viable viral shedding after inoculation by chick embryo inoculation three days after virus challenge (Week 8). While we noted some embryonic lethality at the 0.5 mg dose, there was no embryonic lethality at 5, 50 or 500 mg groups (data not shown).", "While we noted some embryonic lethality at the 0.5 mg dose, there was no embryonic lethality at 5, 50 or 500 mg groups (data not shown). Since the HPAI H5N1 virus first appeared ten years ago, this highly pathogenic avian influenza virus has shown increasing diversification and dissemination in Asia, Africa, and Europe [28, [41] [42] [43] [44] . In addition to its effects on human health by crossspecies transmission [28, 45, 46] and ability to compromise food sources, it poses a continuing threat to public health as it evolves and adapts in different species.", "In addition to its effects on human health by crossspecies transmission [28, 45, 46] and ability to compromise food sources, it poses a continuing threat to public health as it evolves and adapts in different species. The pandemic potential of this virus, especially as it relates to the poultry industry and for reservoir avian hosts, underscores the need for a vaccine that offers broad spectrum immunity and protection against lethal viral challenge. While the virus remains restricted in its ability to infect humans and undergo efficient human-to-human transmission [28, 47] , its persistence and spread in poultry increases the risk of the emergence of a pandemic strain.", "While the virus remains restricted in its ability to infect humans and undergo efficient human-to-human transmission [28, 47] , its persistence and spread in poultry increases the risk of the emergence of a pandemic strain. One approach to pandemic risk reduction is to limit the propagation of the virus in poultry and other relevant avian species. We have previously reported that DNA vaccines encoding HA can confer protection against a highly lethal human pandemic influenza virus, the 1918 H1N1 virus, in mice [36] .", "We have previously reported that DNA vaccines encoding HA can confer protection against a highly lethal human pandemic influenza virus, the 1918 H1N1 virus, in mice [36] . DNA vaccines offer several advantages, including the ability to express diverse antigens, tolerability in various hosts, ease of delivery, and stability for storage and distribution without the necessity of maintaining a cold chain; they have been shown to be safe and efficacious in a variety of animal models [2, 4, 12, 22, 48] . Because they do not contain other viral proteins used to screen for infection, they also address the need to differentiate vaccinated from infected animals.", "Because they do not contain other viral proteins used to screen for infection, they also address the need to differentiate vaccinated from infected animals. There is evidence that DNA vaccination elicits cell-mediated immunity against influenza HA in addition to inducing an antibody response [36] , an effect that could significantly contribute to protective immunity as viruses show genetic drift and reduced susceptibility to neutralization. Ideally, a highly effective influenza vaccine should not only be able to let the host develop a protective immune response against a matching live virus challenge but also elicit robust protective immune responses against a broad range of homologous and heterologous H5 influenza strains.", "Ideally, a highly effective influenza vaccine should not only be able to let the host develop a protective immune response against a matching live virus challenge but also elicit robust protective immune responses against a broad range of homologous and heterologous H5 influenza strains. A multivalent H5 vaccine containing diverse serotypes could expand the antigenic breadth sufficiently to provide protection against heterologous challenge and may preclude the emergence of vaccine-resistant strains that may arise due to evolutionary vaccine pressure on the virus. Due to the antigenic drift and shift of the influenza virus genome, it has been very difficult to predict the next dominant strain of an avian endemic outbreak.", "Due to the antigenic drift and shift of the influenza virus genome, it has been very difficult to predict the next dominant strain of an avian endemic outbreak. DNA vaccines can be synthesized in a relatively short period of time, and the targeted mutations can be tailored to specific viral serotypes. The mutations promote a focused and enhanced immune response [3, 49, 50] that may be particularly important in the event of an outbreak where specificity is the key to epidemic control.", "The mutations promote a focused and enhanced immune response [3, 49, 50] that may be particularly important in the event of an outbreak where specificity is the key to epidemic control. The use of modified codons ensures maximal expression in the host and eliminates the possibility of recombination with influenza viruses that might potentially generate new strains. A more broadly protective murine vaccine was developed here by including more HAs from varying strains in the multivalent vaccine (Figs.", "A more broadly protective murine vaccine was developed here by including more HAs from varying strains in the multivalent vaccine (Figs. 2 and 3) . However, it is less practical to include large numbers of different HAs in one vaccine due to the cost and complexity of manufacturing such a vaccine.", "However, it is less practical to include large numbers of different HAs in one vaccine due to the cost and complexity of manufacturing such a vaccine. Therefore, we simplified the vaccine regimen based on cross-neutralization studies and phylogenetic relationships. A trivalent vaccine was subsequently identified for further studies. Due [51] .", "A trivalent vaccine was subsequently identified for further studies. Due [51] . While three DNA immunizations were used initially to demonstrate protective immunity and have been used previously to elicit protection in mice [36] , we found that effective protective immunity could be induced with two DNA vaccinations and as little as 5 mg trivalent DNA immunization using the ID/SC route with the Agro-JetH device.", "While three DNA immunizations were used initially to demonstrate protective immunity and have been used previously to elicit protection in mice [36] , we found that effective protective immunity could be induced with two DNA vaccinations and as little as 5 mg trivalent DNA immunization using the ID/SC route with the Agro-JetH device. In addition, based on the chick embryo inoculation data, we believe that there is effective neutralization of the virus and lack of infectious viral shedding in chicken vaccinated with as little as 5 mg of DNA. The device's capacity for rapid repetitive injection and the lower quantity and stability of DNA enhance the practicality and utility of this approach for vaccination of endangered species in captivity or administration to poultry or other animals.", "The device's capacity for rapid repetitive injection and the lower quantity and stability of DNA enhance the practicality and utility of this approach for vaccination of endangered species in captivity or administration to poultry or other animals. A/Vietnam/1203/2004 (H5N1) (A/VN/1203/04) was obtained from the repository at the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia. The virus was propagated in 10-day old embryonated chicken eggs at 35uC and stored at 270uC until use.", "The virus was propagated in 10-day old embryonated chicken eggs at 35uC and stored at 270uC until use. The virus was titrated by the Reed and Muench method to determine EID 50 [52] . GenBank ABD28180) were synthesized using human-preferred codons (GeneArt, Regensburg, Germany) [36] .", "GenBank ABD28180) were synthesized using human-preferred codons (GeneArt, Regensburg, Germany) [36] . HA cDNAs from diverse strains of influenza viruses were then inserted into plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo [34, 35, 36] . For initial trivalent immunizations in chickens, the A/Vietnam/1203/ 2004, A/Anhui/1/2005 and A/Indonesia/05/2005 strains were used and in the dose response study, the Vietnam strain was replaced with A/chicken/Nigeria/641/2006.", "For initial trivalent immunizations in chickens, the A/Vietnam/1203/ 2004, A/Anhui/1/2005 and A/Indonesia/05/2005 strains were used and in the dose response study, the Vietnam strain was replaced with A/chicken/Nigeria/641/2006. The immunogens used in DNA vaccination contained a cleavage site mutation (PQRERRRKKRG to PQRETRG) as previously described [35, 36] . This mutation was generated by site-directed mutagenesis using a QuickChange kit (Stratagene, La Jolla, CA).", "This mutation was generated by site-directed mutagenesis using a QuickChange kit (Stratagene, La Jolla, CA). DNA immunization of mice [6] [7] [8] week old female BALB/c mice were purchased from The Jackson Laboratory and maintained in the AAALAC-accredited Vaccine Research Center Animal Care Facility (Bethesda, MD) under specific pathogen-free conditions. All experiments were approved by the Vaccine Research Center Animal Care and Use Committee.", "All experiments were approved by the Vaccine Research Center Animal Care and Use Committee. The mice were immunized as previously described [5] . Briefly, mice (10 animals for all test groups, 20 animals for the\n\nThe study was carried out in the AAALAC-accredited animal facility at the University of Maryland School of Medicine.", "Briefly, mice (10 animals for all test groups, 20 animals for the\n\nThe study was carried out in the AAALAC-accredited animal facility at the University of Maryland School of Medicine. Six groups of 8 one-day-old male and female SPAFAS White Leghorn Chickens, Gallus domesticus, were obtained from Charles River Laboratories (Connecticut). The animals were housed in brooder and grower cages (McMurray Hatcheries, Iowa).", "The animals were housed in brooder and grower cages (McMurray Hatcheries, Iowa). Feed (Teklad Japanese Quail Diet -3050, Harlan-Teklad, WI) and water were provided to the animals ad libitum. The study was performed in strict accordance with the ''Guide'' after approvals from the Animal Care and Use Committees of the Vaccine Research Center, NIH and the University of Maryland.", "The study was performed in strict accordance with the ''Guide'' after approvals from the Animal Care and Use Committees of the Vaccine Research Center, NIH and the University of Maryland. DNA immunizations were performed as described at 0, 3 and 6 weeks. A total dose of 500 mg of one or a combination of the following DNA plasmids in a volume of 250 ml was administered to each animal: pCMV/ R, pCMV/R-HA \n\nAgro-JetH is a needle-free device used for mass delivery of vaccines and drugs in livestock and poultry.", "A total dose of 500 mg of one or a combination of the following DNA plasmids in a volume of 250 ml was administered to each animal: pCMV/ R, pCMV/R-HA \n\nAgro-JetH is a needle-free device used for mass delivery of vaccines and drugs in livestock and poultry. The device is semiautomatic and requires a small CO 2 tank or compressed air for low pressure delivery. Upon trigger activation, CO 2 disperses the injectate at a precise dose into the muscle, dermis or subcutaneous tissue depending on the setting that was standardized for our use.", "Upon trigger activation, CO 2 disperses the injectate at a precise dose into the muscle, dermis or subcutaneous tissue depending on the setting that was standardized for our use. We used an effective volume of 0.1 ml in our injectate [39] . In this study we were able to effectively deliver 0.1 ml of injectate into the animal's dermis/subcutaneous tissue at a pressure of 48-55 psi.", "In this study we were able to effectively deliver 0.1 ml of injectate into the animal's dermis/subcutaneous tissue at a pressure of 48-55 psi. Sixty-eight weeks after the last immunization, female BALB/c mice were lightly anesthetized with Ketamine/Xylazine and inoculated intranasally with 10 LD 50 of A/Vietnam/1203/2004 virus diluted in phosphate-buffered saline in a 50 ul volume. Mice were monitored daily for morbidity and measured for weight loss and mortality for 21 days post infection.", "Mice were monitored daily for morbidity and measured for weight loss and mortality for 21 days post infection. Any mouse that had lost more than 25% of its body weight was euthanized. All experiments involving the HPAI virus were conducted in an AAALAC accredited facility (BioQual Inc., Gaithersburg, MD) under BSL 3 conditions that included enhancements required by the USDA and the Select Agent Program.", "All experiments involving the HPAI virus were conducted in an AAALAC accredited facility (BioQual Inc., Gaithersburg, MD) under BSL 3 conditions that included enhancements required by the USDA and the Select Agent Program. White Leghorn chickens were challenged one week after the last immunization with 20 lethal dose 50 (LD 50 ) of A/Vietnam/1203/04 (H5N1) influenza A virus, equivalent to 2610 4 EID 50 based on previous challenges [53] . Chickens were infected with 200 ml virus intranasally.", "Chickens were infected with 200 ml virus intranasally. Tracheal and cloacal swabs were collected days 3 and 5 post-challenge and stored in glass vials containing BHI medium (BBL TM Brain Heart Infusion, Becton Dickinson) at 280uC. Blood was collected 14 days post-challenge and serum was titered by microneutralization assay.", "Blood was collected 14 days post-challenge and serum was titered by microneutralization assay. Chickens were observed and scored daily for clinical signs of infection, morbidity and mortality. Chickens that survived the study were bled and humanely euthanized at day 14 post-challenge. Lungs, heart, intestine and kidney were collected and samples were stored in formalin for histopathology.", "Lungs, heart, intestine and kidney were collected and samples were stored in formalin for histopathology. Experiments were carried out under BSL3+ conditions with investigators wearing appropriate protective equipment and compliant with all Institutional Animal Care and Use Committee-approved protocols and under Animal Welfare Act regulations at the University of Maryland, College Park, Maryland. Representative tracheal and cloacal swabs were chosen to run an EID 50 assay for comparison and virus titers were determine by the method of Reed and Meunch [52] .", "Representative tracheal and cloacal swabs were chosen to run an EID 50 assay for comparison and virus titers were determine by the method of Reed and Meunch [52] . Briefly, swabs were used to infect 10 day-old embryonated chicken eggs in 10-fold dilutions. Three eggs were inoculated per dilution and incubated for 48 hours before titration.", "Three eggs were inoculated per dilution and incubated for 48 hours before titration. Neutralizing antibodies were titrated from serum samples collected week 5 and 7 post-vaccination and day 14 post-challenge. The microneutralization assay was performed using a 96-well plate format.", "The microneutralization assay was performed using a 96-well plate format. Serum was treated with receptor-destroying enzyme (Denka Seiken Co.) and treated at 37uC per the manufacturer's instructions. After an overnight incubation and subsequent inactivation samples were brought to a final dilution of 1:10 using PBS and each sample was serially diluted and virus, diluted to 100 TCID 50 , was added to each well.", "After an overnight incubation and subsequent inactivation samples were brought to a final dilution of 1:10 using PBS and each sample was serially diluted and virus, diluted to 100 TCID 50 , was added to each well. The plates were then incubated at 37uC, 5% CO 2 for 1-2 hours. Following incubation, supernatants were used to infect a second 96-well plate of MDCK cells.", "Following incubation, supernatants were used to infect a second 96-well plate of MDCK cells. Microplates were incubated at 4uC for 15 minutes and then 37uC, 5% CO 2 for 45 minutes. Supernatants of serum and virus were then discarded and 200 ml of OptiMEM (containing 1X antibiotics/antimycotics, 1 mg/ml TPCK-trypsin) was added and incubated at 37uC, 5% CO 2 for 3 days.", "Supernatants of serum and virus were then discarded and 200 ml of OptiMEM (containing 1X antibiotics/antimycotics, 1 mg/ml TPCK-trypsin) was added and incubated at 37uC, 5% CO 2 for 3 days. After 3 days, 50 ml of the supernatant from each well was transferred into a new 96-well microplate, and an HA assay was performed to calculate the antibody titers. Virus and cell controls were included in the assay.", "Virus and cell controls were included in the assay. Two-fold dilutions of heat-inactivated sera were tested in a microneutralization assay as previously described [54] for the presence of antibodies that neutralized the infectivity of 100 TCID 50 (50% tissue culture infectious dose) of the A/Vietnam/ 1203/2004 H5N1 virus on MDCK cell monolayers by using two wells per dilution on a 96-well plate. The recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described [35, 36] .", "The recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described [35, 36] . For the neutralization assay, antisera from immunized animals were heat-inactivated at 55uC for 30 minutes and mixed with 50 ml of pseudovirus at various dilutions. The sera/virus mixture was then added to 293A cells in 96-well B&W TC Isoplates (Wallac, Turku, Finland; 12,000 cells/well).", "The sera/virus mixture was then added to 293A cells in 96-well B&W TC Isoplates (Wallac, Turku, Finland; 12,000 cells/well). Two hours later, the plates were washed and fresh medium was added. Cells were lysed in mammalian cell lysis buffer (Promega, Madison, WI) 24 hrs after infection and luciferase activity was measured using the Luciferase Assay System (Promega, Madison, WI).", "Cells were lysed in mammalian cell lysis buffer (Promega, Madison, WI) 24 hrs after infection and luciferase activity was measured using the Luciferase Assay System (Promega, Madison, WI). The following strains were used for the production of pseudotyped viruses: for HA we used A/Thailand/1(KAN- \n\nThe HA/HI titers were determined as previously described [54] . Briefly, HA titers were calculated using 50 ml of 0.5% chicken red blood cell suspension in PBS added to 50 ml of twofold dilutions of virus in PBS.", "Briefly, HA titers were calculated using 50 ml of 0.5% chicken red blood cell suspension in PBS added to 50 ml of twofold dilutions of virus in PBS. This mix was incubated at room temperature for 30 minutes. The HA titers were calculated as the reciprocal value of the highest dilution that caused complete hemagglutination.", "The HA titers were calculated as the reciprocal value of the highest dilution that caused complete hemagglutination. HI titers were calculated by titrating 50 ml of antiserum treated with receptor-destroying enzyme and an equivalent amount of A/Vietnam/1203/2004 virus (four hemagglutinating doses) was added to each well. Wells were incubated at room temperature for 30 minutes and 50 ml of a 0.5% suspension of chicken red blood cells was added.", "Wells were incubated at room temperature for 30 minutes and 50 ml of a 0.5% suspension of chicken red blood cells was added. HI titers were calculated after 30 minutes as the reciprocal of the serum dilution that inhibited hemagglutination. Table S1 Hemagglutination inhibition (HI), microneutralization titer (NT), and LAI of sera from individual chickens immunized with different vaccines.", "Table S1 Hemagglutination inhibition (HI), microneutralization titer (NT), and LAI of sera from individual chickens immunized with different vaccines. Sera from immunized animals were obtained at week 5 or 7, a week before or after the final boost, and neutralization was assessed by HI, microneutralization (NT) and LAI (shown as IC 50 ). Individual animal serum of each group is shown and was analyzed as described in the Materials and Methods section.", "Individual animal serum of each group is shown and was analyzed as described in the Materials and Methods section. Figure S1 Characterization of needle-free (Agro-JetH) DNA immunization in chickens. To evaluate the distribution of fluid into superficial or deep layers of subcutaneous tissues after delivery by AgroJetH, 4 or 7 week old chickens were injected with a solution containing India ink with this needle-free device at various pressures, ranging from 45 to 55 mm Hg.", "To evaluate the distribution of fluid into superficial or deep layers of subcutaneous tissues after delivery by AgroJetH, 4 or 7 week old chickens were injected with a solution containing India ink with this needle-free device at various pressures, ranging from 45 to 55 mm Hg. Three sites (thigh, wing and breast) were used, and biopsies were taken for routine hematoxylin and eosin staining. Representative sections of thigh injections are shown from 7 week old chickens and were similar at 4 weeks (data not shown).", "Representative sections of thigh injections are shown from 7 week old chickens and were similar at 4 weeks (data not shown). While the 48 mm Hg pressure deposited the injectate into the dermis/subcutaneous region (left), the higher pressure injections, 52 and 58 mm Hg, deposited the injectate into the subcutaneous and muscle layers (middle, right). 48 mm Hg consistently provided an optimal pressure to deposit the injectate into the dermis and subcutaneous tissue and was chosen for all AgroJetH immunizations.", "48 mm Hg consistently provided an optimal pressure to deposit the injectate into the dermis and subcutaneous tissue and was chosen for all AgroJetH immunizations. Found at: .1371/journal.pone.0002432.s003 (10.74 MB DOC)" ]

[ "BACKGROUND: Sustained outbreaks of highly pathogenic avian influenza (HPAI) H5N1 in avian species increase the risk of reassortment and adaptation to humans. The ability to contain its spread in chickens would reduce this threat and help maintain the capacity for egg-based vaccine production. While vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses.", "While vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses. METHODOLOGY / PRINCIPAL FINDINGS: The ability of DNA vaccines encoding hemagglutinin (HA) proteins from different HPAI H5N1 serotypes was evaluated for its ability to elicit neutralizing antibodies and to protect against homologous and heterologous HPAI H5N1 strain challenge in mice and chickens after DNA immunization by needle and syringe or with a pressure injection device. These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs.", "These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs. The response was dose-dependent, and breadth was determined by the choice of the influenza virus HA in the vaccine. Monovalent and trivalent HA vaccines were tested first in mice and conferred protection against lethal H5N1 A/Vietnam/1203/2004 challenge 68 weeks after vaccination.", "Monovalent and trivalent HA vaccines were tested first in mice and conferred protection against lethal H5N1 A/Vietnam/1203/2004 challenge 68 weeks after vaccination. In chickens, protection was observed against heterologous strains of HPAI H5N1 after vaccination with a trivalent H5 serotype DNA vaccine with doses as low as 5 µg DNA given twice either by intramuscular needle injection or with a needle-free device. CONCLUSIONS/SIGNIFICANCE: DNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species.", "CONCLUSIONS/SIGNIFICANCE: DNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. In addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates. Text: The highly pathogenic H5N1 influenza virus causes lethal multi-organ disease in poultry, resulting in significant economic losses and a public health concern in many parts of the world.", "Text: The highly pathogenic H5N1 influenza virus causes lethal multi-organ disease in poultry, resulting in significant economic losses and a public health concern in many parts of the world. The greatest threats posed by this virus are its ability to cause mortality in humans, its potential to compromise food supplies, and its possible economic impacts. Viral maintenance in poultry potentiates the risk of human-to-human transmission and the emergence of a pandemic strain through reassortment.", "Viral maintenance in poultry potentiates the risk of human-to-human transmission and the emergence of a pandemic strain through reassortment. An effective, safe poultry vaccine that elicits broadly protective immune responses to evolving flu strains would provide a countermeasure to reduce the likelihood of transmission of this virus from domestic birds to humans and simultaneously would protect commercial poultry operations and subsistence farmers. DNA vaccines have been shown to elicit robust immune responses in various animal species, from mice to nonhuman primates [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] .", "DNA vaccines have been shown to elicit robust immune responses in various animal species, from mice to nonhuman primates [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] . In human trials, these vaccines elicit cellular and humoral immune responses against various infectious agents, including influenza, SARS, SIV and HIV. In addition to their ability to elicit antibody responses, they also stimulate antigenspecific and sustained T cell responses [1] [2] [3] 6, 12, 13] .", "In addition to their ability to elicit antibody responses, they also stimulate antigenspecific and sustained T cell responses [1] [2] [3] 6, 12, 13] . DNA vaccination has been used experimentally against various infectious agents in a variety of mammals, including cattle (against infectious bovine rhinotracheitis/bovine diarrhea virus, leptospirosis and mycobacteriosis) [14, 15] , pigs (against classical swine fever virus and mycoplasmosis) [16] , and horses (against West Nile virus and rabies) [17] . In addition, DNA vaccines have been tested against avian plasmodium infection in penguins [18] and against influenza and infectious bursal disease in chickens [7, 8, 19] , duck hepatitis B virus in ducks [6] , and avian metapneumovirus and Chlamydia psittaci in turkeys [20, 21] (reviewed in ref.", "In addition, DNA vaccines have been tested against avian plasmodium infection in penguins [18] and against influenza and infectious bursal disease in chickens [7, 8, 19] , duck hepatitis B virus in ducks [6] , and avian metapneumovirus and Chlamydia psittaci in turkeys [20, 21] (reviewed in ref. [22] ). While they have been used in chickens to generate antisera to specific influenza viruses and confer protection against the low pathogenicity H5N2 strain [23] , there is only one previous report of a monovalent DNA vaccine effective against H5N1 (and that only against a matched H5N1 isolate) [24] ; no protection with multivalent DNA vaccines against heterologous strains has been reported.", "While they have been used in chickens to generate antisera to specific influenza viruses and confer protection against the low pathogenicity H5N2 strain [23] , there is only one previous report of a monovalent DNA vaccine effective against H5N1 (and that only against a matched H5N1 isolate) [24] ; no protection with multivalent DNA vaccines against heterologous strains has been reported. Development and characterization of a DNA vaccine modality for use in poultry offers a potential countermeasure against HPAI H5N1 avian influenza outbreaks. The virus can infect humans, typically from animal sources, including commercial and wild avian species, livestock, and possibly other non-domesticated animal species [25] [26] [27] .", "The virus can infect humans, typically from animal sources, including commercial and wild avian species, livestock, and possibly other non-domesticated animal species [25] [26] [27] . While there is marked diversity in the host range of type A influenza viruses, many experts have speculated that a pandemic strain of type A influenza could evolve in avian species or avian influenza viruses could contribute virulent genes to a pandemic strain through reassortment [28, 29] . Thus, there is reason to consider vaccination of poultry that would stimulate potent and broad protective immune responses [7, 30, 31] .", "Thus, there is reason to consider vaccination of poultry that would stimulate potent and broad protective immune responses [7, 30, 31] . In undertaking such efforts, it is important that there be a differentiation of infected from vaccinated animals [32] so that animals can be protected and permit monitoring of new infections using proven and sensitive methodologies. In this study, we used an automated high capacity needle-free injection device, Agro-JetH (Medical International Technology, Inc., Denver, CO) to explore the feasibility of DNA vaccination of poultry.", "In this study, we used an automated high capacity needle-free injection device, Agro-JetH (Medical International Technology, Inc., Denver, CO) to explore the feasibility of DNA vaccination of poultry. After optimization of injection conditions, alternative multivalent DNA vaccine regimens were analyzed and compared for magnitude and breadth of neutralizing antibodies, as well as protective efficacy after challenge in mouse and chicken models of HPAI H5N1 infection. The findings suggest that it is possible to develop a multivalent DNA vaccine for poultry that can protect against multiple HPAI H5N1 strains and that could keep pace with the continued evolution of avian influenza viruses.", "The findings suggest that it is possible to develop a multivalent DNA vaccine for poultry that can protect against multiple HPAI H5N1 strains and that could keep pace with the continued evolution of avian influenza viruses. Immunogenicity and neutralizing antibody specificity of alternative HA DNA vaccines in mice To evaluate the efficacy of multivalent DNA vaccines, initial studies were performed in mice. Expression vectors encoding HAs from ten phylogenetically diverse strains of influenza viruses [33] were generated by synthesis of cDNAs (see Materials and Methods) in plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo [34, 35, 36] .", "Expression vectors encoding HAs from ten phylogenetically diverse strains of influenza viruses [33] were generated by synthesis of cDNAs (see Materials and Methods) in plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo [34, 35, 36] . Animals were immunized with each expression vector intramuscularly (IM) at three week intervals, and the antisera were evaluated on day 14 after the third immunization for their ability to neutralize HPAI H5N1 pseudotyped lentiviral vectors as previously described [35, 36] . We have previously shown that lentiviral assay inhibition (LAI) yields similar results to microneutralization and HAI analyses with higher sensitivity in mice [35, 36] \n\nTo determine whether immunization with multiple HAs simultaneously could expand the breadth of the neutralizing antibody response without significant loss of magnitude, a combination of 10 HA DNA vaccine immunogens was administered IM at proportionally lower concentration (1.5 mg per immunogen) into groups of 10 mice (see Materials and Methods).", "We have previously shown that lentiviral assay inhibition (LAI) yields similar results to microneutralization and HAI analyses with higher sensitivity in mice [35, 36] \n\nTo determine whether immunization with multiple HAs simultaneously could expand the breadth of the neutralizing antibody response without significant loss of magnitude, a combination of 10 HA DNA vaccine immunogens was administered IM at proportionally lower concentration (1.5 mg per immunogen) into groups of 10 mice (see Materials and Methods). Remarkably, despite a log lower DNA concentration of each component, significant neutralizing antibody titers were generated to each of the 10 immunogens, with .80% neutralization against 6 out of 12 H5 HA pseudoviruses at dilutions of up to 1:400 ( Fig. 2A) .", "2A) . To evaluate whether similar breadth of immunity could be generated with fewer immunogens, two different combinations of 5 immunogens were selected, based on the phylogenetic diversity of HA among the avian influenza viruses [33] and the crossreactivity of the neutralizing antibody responses of select individual immunogens (Fig. 1 ).", "1 ). As expected, there were substantial differences in the breadth of neutralization between these two sets of 5 immunogen multivalent vaccines (Fig. 2 , B vs. C).", "2 , B vs. C). In one set, while neutralization of homologous strains was comparable to the monovalent and the 10 immunogen multivalent immune response, fewer cross-reactive antibodies were detected, directed most prominently against A/Iraq Protection of DNA-vaccinated mice against challenge with heterologous H5N1 A/Vietnam/1203/2004 influenza virus Mice immunized as described above were challenged with a heterologous H5N1 virus 68 weeks after the final DNA vaccination. Animals were then challenged with 10 LD 50 of the highly pathogenic A/Vietnam/1203/2004 virus intranasally, and morbidity and mortality were monitored for 21 days after the viral challenge.", "Animals were then challenged with 10 LD 50 of the highly pathogenic A/Vietnam/1203/2004 virus intranasally, and morbidity and mortality were monitored for 21 days after the viral challenge. The control animals, injected with the plasmid expression vector with no insert, died within 10 days of infection. Complete survival was observed in the groups immunized with the 10 component and set 2 of the 5 component multivalent DNA vaccines (Fig.", "Complete survival was observed in the groups immunized with the 10 component and set 2 of the 5 component multivalent DNA vaccines (Fig. 3) . Immunization with HA derived from the A/ Indonesia/05/2005 strain or set 1 of the 5 component multivalent DNA vaccine showed a survival rate approaching 90%.", "Immunization with HA derived from the A/ Indonesia/05/2005 strain or set 1 of the 5 component multivalent DNA vaccine showed a survival rate approaching 90%. In contrast, animals injected with HA plasmid DNA derived from A/ Anhui/1/2005, which has diverged more from A/Vietnam/ 1203/2004, showed a lower percent survival (70%) after lethal viral challenge. Survival differences between groups were assessed using a log-rank test and the Gehan-Wilcoxon test on the survival curves for pairs of groups.", "Survival differences between groups were assessed using a log-rank test and the Gehan-Wilcoxon test on the survival curves for pairs of groups. A test was deemed significant if the pvalue was ,0.01. Mice injected IM with different HAs, A/ Indonesia/5/05, A/Anhui/1/05, 10HA, 5 HA (Set 1), or 5 HA (Set 2) showed a significant difference compared to control (all p values,0.001).", "Mice injected IM with different HAs, A/ Indonesia/5/05, A/Anhui/1/05, 10HA, 5 HA (Set 1), or 5 HA (Set 2) showed a significant difference compared to control (all p values,0.001). Among the HA-immunized groups, there was no significant difference between any two groups (p.0.08 for all comparisons). For example, no significant difference was observed between the A/Anhui/1/05 group, which had the least survival among the HA immunized groups (7 out of 10), and other HA groups: A/Indonesia/5/05 (p = 0.377), 10 HA (p = 0.082), 5 HA (Set 1) (p = 0.101), or 5 HA (Set 2) (p = .411).", "For example, no significant difference was observed between the A/Anhui/1/05 group, which had the least survival among the HA immunized groups (7 out of 10), and other HA groups: A/Indonesia/5/05 (p = 0.377), 10 HA (p = 0.082), 5 HA (Set 1) (p = 0.101), or 5 HA (Set 2) (p = .411). Therefore, we cannot exclude the possibility that the 3 deaths in the A/Anhui/1/05 group may have been due to random chance. Since it is desirable to confer protective immunity in poultry and HA DNA vaccination was effective in mice, we next examined the breadth and potency of single or multiple HA plasmid immunization in chickens.", "Since it is desirable to confer protective immunity in poultry and HA DNA vaccination was effective in mice, we next examined the breadth and potency of single or multiple HA plasmid immunization in chickens. The ability of chickens to generate specific antibodies was assessed with three strains that showed broad cross protection in mouse studies (A/Vietnam/1203/2004, A/Anhui/ 1/2005 and A/Indonesia/05/2005), administered individually or in combination, by different injection methods. In addition to needle injection, a needle-free repetitive injection device, Agro-JetH (Medical International Technology, Inc., Denver, CO), was analyzed.", "In addition to needle injection, a needle-free repetitive injection device, Agro-JetH (Medical International Technology, Inc., Denver, CO), was analyzed. This device disperses the 0.1 to 5 ml injection doses into the dermal, subcutaneous, or intramuscular tissue depending upon the pressure adjustments, powered by a CO 2 gas pressure plunger [39] . The injection conditions were determined by histologic analysis of tissues that received injections of India ink; a pressure of 48 psi was chosen since it enabled consistent delivery into intradermal and subcutaneous tissues (Fig.", "The injection conditions were determined by histologic analysis of tissues that received injections of India ink; a pressure of 48 psi was chosen since it enabled consistent delivery into intradermal and subcutaneous tissues (Fig. S1 ). Immunization of chickens with the control plasmid (CMV/R) without an HA gene insert elicited minimal neutralizing antibody titers compared to HA-immunized animals 1 week after 3 DNA immunizations.", "Immunization of chickens with the control plasmid (CMV/R) without an HA gene insert elicited minimal neutralizing antibody titers compared to HA-immunized animals 1 week after 3 DNA immunizations. Nearly all chickens immunized with either monovalent or multivalent HA DNA vaccines generated significant neutralization titers ( Fig. 4 and Table S1 ).", "4 and Table S1 ). In general, there was a progressive increase in the amount of neutralization after each successive DNA vaccination (data not shown) with maximal response at 1 week after the 3 rd DNA immunization, with highest and most consistent levels in the trivalent vaccine group delivered with the Agro-JetH device. Neutralization of the Indonesia HA strain was the most robust, with neutralization nearing 100% at titers greater than 1:3200.", "Neutralization of the Indonesia HA strain was the most robust, with neutralization nearing 100% at titers greater than 1:3200. Both the monovalent and multivalent vaccines elicited robust homologous ( vaccine (Fig. 4 ).", "4 ). Even though one chicken (238) in the multivalent vaccine group produced almost the same degree of neutralization at each time point and was protected, it did not produce a high neutralizing antibody titer for reasons that were uncertain but possibly related to a non-specific inhibitor in the sera. To determine whether chickens immunized with single or multiple DNA vaccines were protected from a lethal challenge of a heterologous HPAI H5N1 virus, vaccinated chickens were In panels B and C, mice (n = 10) were immunized with 15 mg of plasmid (3 mg each) three times at 3 week intervals.", "To determine whether chickens immunized with single or multiple DNA vaccines were protected from a lethal challenge of a heterologous HPAI H5N1 virus, vaccinated chickens were In panels B and C, mice (n = 10) were immunized with 15 mg of plasmid (3 mg each) three times at 3 week intervals. Serum pools from the immunized animals were collected 14 days after the third immunization. The antisera were tested against the 12 indicated pseudotyped lentiviral vectors at varying dilutions.", "The antisera were tested against the 12 indicated pseudotyped lentiviral vectors at varying dilutions. Error bars at each point indicate the standard deviation; each sample was evaluated in triplicate. In general, the immunized serum neutralized all tested pseudotyped lentiviruses at low dilutions while differences were often observed at high dilution.", "In general, the immunized serum neutralized all tested pseudotyped lentiviruses at low dilutions while differences were often observed at high dilution. .1371/journal.pone.0002432.g002 inoculated with 20 LD 50 of highly pathogenic A/Vietnam/1203/ 2004 heterologous virus intranasally using standard methods [25, 40] and monitored for morbidity, mortality, viral shedding and serum antibodies. While all the control animals died within 2 days of infection, 100% survival was noted in the rest of the chickens (Fig.", "While all the control animals died within 2 days of infection, 100% survival was noted in the rest of the chickens (Fig. 5A ). The animals that were healthy, showing no signs of clinical disease or malaise, were euthanized on day 14.", "The animals that were healthy, showing no signs of clinical disease or malaise, were euthanized on day 14. There was no evidence for viral shedding monitored via tracheal and cloacal swabs of infected chickens 2-14 days after challenge as determined by embryonal inoculation (data not shown: egg infectious dose 50 (EID 50 ) limit of detection ,100 virus particles). To compare the relative efficacy of DNA vaccines delivered IM by needle and syringe versus the needle-free Agro-JetH device injection, a dose-response study was performed with amounts of DNA vaccine ranging from 500 to 0.5 mg with two inoculations.", "To compare the relative efficacy of DNA vaccines delivered IM by needle and syringe versus the needle-free Agro-JetH device injection, a dose-response study was performed with amounts of DNA vaccine ranging from 500 to 0.5 mg with two inoculations. In these experiments, the HA derived from A/chicken/Nigeria/641/ 2006 was substituted for A/Vietnam/1203/2004 since it represented a more contemporary isolate. The observed rate of protection was higher among the animals receiving 5 mg by Agro-Jet (8/8) than by IM injection (6/8) (Fig.", "The observed rate of protection was higher among the animals receiving 5 mg by Agro-Jet (8/8) than by IM injection (6/8) (Fig. 5, B vs. C). Both modes provided complete protection for all animals at doses higher than this, and 25% protection for the animals receiving 0.5 mg doses (Fig.", "Both modes provided complete protection for all animals at doses higher than this, and 25% protection for the animals receiving 0.5 mg doses (Fig. 5B, C) . Survival differences between consecutive doses were assessed using a log-rank test on the survival curves for pairs of groups.", "Survival differences between consecutive doses were assessed using a log-rank test on the survival curves for pairs of groups. A test was deemed significant if the p-value was ,.01, and marginally significant if the p-value was ,.05 but ..01. Chickens injected IM showed a marginally significant difference between 0.5 and 5 mg (p = .047).", "Chickens injected IM showed a marginally significant difference between 0.5 and 5 mg (p = .047). In the same group there was a significant difference between control and 5, 50 and 500 mg (p,.001 for all comparisons) and the difference between control and 0.5 mg was marginally significant (p = .016). Chickens that were injected using Agro-JetH showed a significant difference between 0.5 and 5 mg (p = .004) and between control and 5, 50, and 500 mg (p,.001 for all comparisons).", "Chickens that were injected using Agro-JetH showed a significant difference between 0.5 and 5 mg (p = .004) and between control and 5, 50, and 500 mg (p,.001 for all comparisons). There were no differences between control and 0.5 mg or between 5, 50, and 500 mg. Lastly, the survival differences between Agro-JetH and IM for each dose group were not significant. The neutralizing antibody response to homologous and heterologous HAs corresponded with protection and correlated with dose, with higher titers elicited by injection with Agro-JetH compared to needle (Table S2) .", "The neutralizing antibody response to homologous and heterologous HAs corresponded with protection and correlated with dose, with higher titers elicited by injection with Agro-JetH compared to needle (Table S2) . We assessed viable viral shedding after inoculation by chick embryo inoculation three days after virus challenge (Week 8). While we noted some embryonic lethality at the 0.5 mg dose, there was no embryonic lethality at 5, 50 or 500 mg groups (data not shown).", "While we noted some embryonic lethality at the 0.5 mg dose, there was no embryonic lethality at 5, 50 or 500 mg groups (data not shown). Since the HPAI H5N1 virus first appeared ten years ago, this highly pathogenic avian influenza virus has shown increasing diversification and dissemination in Asia, Africa, and Europe [28, [41] [42] [43] [44] . In addition to its effects on human health by crossspecies transmission [28, 45, 46] and ability to compromise food sources, it poses a continuing threat to public health as it evolves and adapts in different species.", "In addition to its effects on human health by crossspecies transmission [28, 45, 46] and ability to compromise food sources, it poses a continuing threat to public health as it evolves and adapts in different species. The pandemic potential of this virus, especially as it relates to the poultry industry and for reservoir avian hosts, underscores the need for a vaccine that offers broad spectrum immunity and protection against lethal viral challenge. While the virus remains restricted in its ability to infect humans and undergo efficient human-to-human transmission [28, 47] , its persistence and spread in poultry increases the risk of the emergence of a pandemic strain.", "While the virus remains restricted in its ability to infect humans and undergo efficient human-to-human transmission [28, 47] , its persistence and spread in poultry increases the risk of the emergence of a pandemic strain. One approach to pandemic risk reduction is to limit the propagation of the virus in poultry and other relevant avian species. We have previously reported that DNA vaccines encoding HA can confer protection against a highly lethal human pandemic influenza virus, the 1918 H1N1 virus, in mice [36] .", "We have previously reported that DNA vaccines encoding HA can confer protection against a highly lethal human pandemic influenza virus, the 1918 H1N1 virus, in mice [36] . DNA vaccines offer several advantages, including the ability to express diverse antigens, tolerability in various hosts, ease of delivery, and stability for storage and distribution without the necessity of maintaining a cold chain; they have been shown to be safe and efficacious in a variety of animal models [2, 4, 12, 22, 48] . Because they do not contain other viral proteins used to screen for infection, they also address the need to differentiate vaccinated from infected animals.", "Because they do not contain other viral proteins used to screen for infection, they also address the need to differentiate vaccinated from infected animals. There is evidence that DNA vaccination elicits cell-mediated immunity against influenza HA in addition to inducing an antibody response [36] , an effect that could significantly contribute to protective immunity as viruses show genetic drift and reduced susceptibility to neutralization. Ideally, a highly effective influenza vaccine should not only be able to let the host develop a protective immune response against a matching live virus challenge but also elicit robust protective immune responses against a broad range of homologous and heterologous H5 influenza strains.", "Ideally, a highly effective influenza vaccine should not only be able to let the host develop a protective immune response against a matching live virus challenge but also elicit robust protective immune responses against a broad range of homologous and heterologous H5 influenza strains. A multivalent H5 vaccine containing diverse serotypes could expand the antigenic breadth sufficiently to provide protection against heterologous challenge and may preclude the emergence of vaccine-resistant strains that may arise due to evolutionary vaccine pressure on the virus. Due to the antigenic drift and shift of the influenza virus genome, it has been very difficult to predict the next dominant strain of an avian endemic outbreak.", "Due to the antigenic drift and shift of the influenza virus genome, it has been very difficult to predict the next dominant strain of an avian endemic outbreak. DNA vaccines can be synthesized in a relatively short period of time, and the targeted mutations can be tailored to specific viral serotypes. The mutations promote a focused and enhanced immune response [3, 49, 50] that may be particularly important in the event of an outbreak where specificity is the key to epidemic control.", "The mutations promote a focused and enhanced immune response [3, 49, 50] that may be particularly important in the event of an outbreak where specificity is the key to epidemic control. The use of modified codons ensures maximal expression in the host and eliminates the possibility of recombination with influenza viruses that might potentially generate new strains. A more broadly protective murine vaccine was developed here by including more HAs from varying strains in the multivalent vaccine (Figs.", "A more broadly protective murine vaccine was developed here by including more HAs from varying strains in the multivalent vaccine (Figs. 2 and 3) . However, it is less practical to include large numbers of different HAs in one vaccine due to the cost and complexity of manufacturing such a vaccine.", "However, it is less practical to include large numbers of different HAs in one vaccine due to the cost and complexity of manufacturing such a vaccine. Therefore, we simplified the vaccine regimen based on cross-neutralization studies and phylogenetic relationships. A trivalent vaccine was subsequently identified for further studies. Due [51] .", "A trivalent vaccine was subsequently identified for further studies. Due [51] . While three DNA immunizations were used initially to demonstrate protective immunity and have been used previously to elicit protection in mice [36] , we found that effective protective immunity could be induced with two DNA vaccinations and as little as 5 mg trivalent DNA immunization using the ID/SC route with the Agro-JetH device.", "While three DNA immunizations were used initially to demonstrate protective immunity and have been used previously to elicit protection in mice [36] , we found that effective protective immunity could be induced with two DNA vaccinations and as little as 5 mg trivalent DNA immunization using the ID/SC route with the Agro-JetH device. In addition, based on the chick embryo inoculation data, we believe that there is effective neutralization of the virus and lack of infectious viral shedding in chicken vaccinated with as little as 5 mg of DNA. The device's capacity for rapid repetitive injection and the lower quantity and stability of DNA enhance the practicality and utility of this approach for vaccination of endangered species in captivity or administration to poultry or other animals.", "The device's capacity for rapid repetitive injection and the lower quantity and stability of DNA enhance the practicality and utility of this approach for vaccination of endangered species in captivity or administration to poultry or other animals. A/Vietnam/1203/2004 (H5N1) (A/VN/1203/04) was obtained from the repository at the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia. The virus was propagated in 10-day old embryonated chicken eggs at 35uC and stored at 270uC until use.", "The virus was propagated in 10-day old embryonated chicken eggs at 35uC and stored at 270uC until use. The virus was titrated by the Reed and Muench method to determine EID 50 [52] . GenBank ABD28180) were synthesized using human-preferred codons (GeneArt, Regensburg, Germany) [36] .", "GenBank ABD28180) were synthesized using human-preferred codons (GeneArt, Regensburg, Germany) [36] . HA cDNAs from diverse strains of influenza viruses were then inserted into plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo [34, 35, 36] . For initial trivalent immunizations in chickens, the A/Vietnam/1203/ 2004, A/Anhui/1/2005 and A/Indonesia/05/2005 strains were used and in the dose response study, the Vietnam strain was replaced with A/chicken/Nigeria/641/2006.", "For initial trivalent immunizations in chickens, the A/Vietnam/1203/ 2004, A/Anhui/1/2005 and A/Indonesia/05/2005 strains were used and in the dose response study, the Vietnam strain was replaced with A/chicken/Nigeria/641/2006. The immunogens used in DNA vaccination contained a cleavage site mutation (PQRERRRKKRG to PQRETRG) as previously described [35, 36] . This mutation was generated by site-directed mutagenesis using a QuickChange kit (Stratagene, La Jolla, CA).", "This mutation was generated by site-directed mutagenesis using a QuickChange kit (Stratagene, La Jolla, CA). DNA immunization of mice [6] [7] [8] week old female BALB/c mice were purchased from The Jackson Laboratory and maintained in the AAALAC-accredited Vaccine Research Center Animal Care Facility (Bethesda, MD) under specific pathogen-free conditions. All experiments were approved by the Vaccine Research Center Animal Care and Use Committee.", "All experiments were approved by the Vaccine Research Center Animal Care and Use Committee. The mice were immunized as previously described [5] . Briefly, mice (10 animals for all test groups, 20 animals for the\n\nThe study was carried out in the AAALAC-accredited animal facility at the University of Maryland School of Medicine.", "Briefly, mice (10 animals for all test groups, 20 animals for the\n\nThe study was carried out in the AAALAC-accredited animal facility at the University of Maryland School of Medicine. Six groups of 8 one-day-old male and female SPAFAS White Leghorn Chickens, Gallus domesticus, were obtained from Charles River Laboratories (Connecticut). The animals were housed in brooder and grower cages (McMurray Hatcheries, Iowa).", "The animals were housed in brooder and grower cages (McMurray Hatcheries, Iowa). Feed (Teklad Japanese Quail Diet -3050, Harlan-Teklad, WI) and water were provided to the animals ad libitum. The study was performed in strict accordance with the ''Guide'' after approvals from the Animal Care and Use Committees of the Vaccine Research Center, NIH and the University of Maryland.", "The study was performed in strict accordance with the ''Guide'' after approvals from the Animal Care and Use Committees of the Vaccine Research Center, NIH and the University of Maryland. DNA immunizations were performed as described at 0, 3 and 6 weeks. A total dose of 500 mg of one or a combination of the following DNA plasmids in a volume of 250 ml was administered to each animal: pCMV/ R, pCMV/R-HA \n\nAgro-JetH is a needle-free device used for mass delivery of vaccines and drugs in livestock and poultry.", "A total dose of 500 mg of one or a combination of the following DNA plasmids in a volume of 250 ml was administered to each animal: pCMV/ R, pCMV/R-HA \n\nAgro-JetH is a needle-free device used for mass delivery of vaccines and drugs in livestock and poultry. The device is semiautomatic and requires a small CO 2 tank or compressed air for low pressure delivery. Upon trigger activation, CO 2 disperses the injectate at a precise dose into the muscle, dermis or subcutaneous tissue depending on the setting that was standardized for our use.", "Upon trigger activation, CO 2 disperses the injectate at a precise dose into the muscle, dermis or subcutaneous tissue depending on the setting that was standardized for our use. We used an effective volume of 0.1 ml in our injectate [39] . In this study we were able to effectively deliver 0.1 ml of injectate into the animal's dermis/subcutaneous tissue at a pressure of 48-55 psi.", "In this study we were able to effectively deliver 0.1 ml of injectate into the animal's dermis/subcutaneous tissue at a pressure of 48-55 psi. Sixty-eight weeks after the last immunization, female BALB/c mice were lightly anesthetized with Ketamine/Xylazine and inoculated intranasally with 10 LD 50 of A/Vietnam/1203/2004 virus diluted in phosphate-buffered saline in a 50 ul volume. Mice were monitored daily for morbidity and measured for weight loss and mortality for 21 days post infection.", "Mice were monitored daily for morbidity and measured for weight loss and mortality for 21 days post infection. Any mouse that had lost more than 25% of its body weight was euthanized. All experiments involving the HPAI virus were conducted in an AAALAC accredited facility (BioQual Inc., Gaithersburg, MD) under BSL 3 conditions that included enhancements required by the USDA and the Select Agent Program.", "All experiments involving the HPAI virus were conducted in an AAALAC accredited facility (BioQual Inc., Gaithersburg, MD) under BSL 3 conditions that included enhancements required by the USDA and the Select Agent Program. White Leghorn chickens were challenged one week after the last immunization with 20 lethal dose 50 (LD 50 ) of A/Vietnam/1203/04 (H5N1) influenza A virus, equivalent to 2610 4 EID 50 based on previous challenges [53] . Chickens were infected with 200 ml virus intranasally.", "Chickens were infected with 200 ml virus intranasally. Tracheal and cloacal swabs were collected days 3 and 5 post-challenge and stored in glass vials containing BHI medium (BBL TM Brain Heart Infusion, Becton Dickinson) at 280uC. Blood was collected 14 days post-challenge and serum was titered by microneutralization assay.", "Blood was collected 14 days post-challenge and serum was titered by microneutralization assay. Chickens were observed and scored daily for clinical signs of infection, morbidity and mortality. Chickens that survived the study were bled and humanely euthanized at day 14 post-challenge. Lungs, heart, intestine and kidney were collected and samples were stored in formalin for histopathology.", "Lungs, heart, intestine and kidney were collected and samples were stored in formalin for histopathology. Experiments were carried out under BSL3+ conditions with investigators wearing appropriate protective equipment and compliant with all Institutional Animal Care and Use Committee-approved protocols and under Animal Welfare Act regulations at the University of Maryland, College Park, Maryland. Representative tracheal and cloacal swabs were chosen to run an EID 50 assay for comparison and virus titers were determine by the method of Reed and Meunch [52] .", "Representative tracheal and cloacal swabs were chosen to run an EID 50 assay for comparison and virus titers were determine by the method of Reed and Meunch [52] . Briefly, swabs were used to infect 10 day-old embryonated chicken eggs in 10-fold dilutions. Three eggs were inoculated per dilution and incubated for 48 hours before titration.", "Three eggs were inoculated per dilution and incubated for 48 hours before titration. Neutralizing antibodies were titrated from serum samples collected week 5 and 7 post-vaccination and day 14 post-challenge. The microneutralization assay was performed using a 96-well plate format.", "The microneutralization assay was performed using a 96-well plate format. Serum was treated with receptor-destroying enzyme (Denka Seiken Co.) and treated at 37uC per the manufacturer's instructions. After an overnight incubation and subsequent inactivation samples were brought to a final dilution of 1:10 using PBS and each sample was serially diluted and virus, diluted to 100 TCID 50 , was added to each well.", "After an overnight incubation and subsequent inactivation samples were brought to a final dilution of 1:10 using PBS and each sample was serially diluted and virus, diluted to 100 TCID 50 , was added to each well. The plates were then incubated at 37uC, 5% CO 2 for 1-2 hours. Following incubation, supernatants were used to infect a second 96-well plate of MDCK cells.", "Following incubation, supernatants were used to infect a second 96-well plate of MDCK cells. Microplates were incubated at 4uC for 15 minutes and then 37uC, 5% CO 2 for 45 minutes. Supernatants of serum and virus were then discarded and 200 ml of OptiMEM (containing 1X antibiotics/antimycotics, 1 mg/ml TPCK-trypsin) was added and incubated at 37uC, 5% CO 2 for 3 days.", "Supernatants of serum and virus were then discarded and 200 ml of OptiMEM (containing 1X antibiotics/antimycotics, 1 mg/ml TPCK-trypsin) was added and incubated at 37uC, 5% CO 2 for 3 days. After 3 days, 50 ml of the supernatant from each well was transferred into a new 96-well microplate, and an HA assay was performed to calculate the antibody titers. Virus and cell controls were included in the assay.", "Virus and cell controls were included in the assay. Two-fold dilutions of heat-inactivated sera were tested in a microneutralization assay as previously described [54] for the presence of antibodies that neutralized the infectivity of 100 TCID 50 (50% tissue culture infectious dose) of the A/Vietnam/ 1203/2004 H5N1 virus on MDCK cell monolayers by using two wells per dilution on a 96-well plate. The recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described [35, 36] .", "The recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described [35, 36] . For the neutralization assay, antisera from immunized animals were heat-inactivated at 55uC for 30 minutes and mixed with 50 ml of pseudovirus at various dilutions. The sera/virus mixture was then added to 293A cells in 96-well B&W TC Isoplates (Wallac, Turku, Finland; 12,000 cells/well).", "The sera/virus mixture was then added to 293A cells in 96-well B&W TC Isoplates (Wallac, Turku, Finland; 12,000 cells/well). Two hours later, the plates were washed and fresh medium was added. Cells were lysed in mammalian cell lysis buffer (Promega, Madison, WI) 24 hrs after infection and luciferase activity was measured using the Luciferase Assay System (Promega, Madison, WI).", "Cells were lysed in mammalian cell lysis buffer (Promega, Madison, WI) 24 hrs after infection and luciferase activity was measured using the Luciferase Assay System (Promega, Madison, WI). The following strains were used for the production of pseudotyped viruses: for HA we used A/Thailand/1(KAN- \n\nThe HA/HI titers were determined as previously described [54] . Briefly, HA titers were calculated using 50 ml of 0.5% chicken red blood cell suspension in PBS added to 50 ml of twofold dilutions of virus in PBS.", "Briefly, HA titers were calculated using 50 ml of 0.5% chicken red blood cell suspension in PBS added to 50 ml of twofold dilutions of virus in PBS. This mix was incubated at room temperature for 30 minutes. The HA titers were calculated as the reciprocal value of the highest dilution that caused complete hemagglutination.", "The HA titers were calculated as the reciprocal value of the highest dilution that caused complete hemagglutination. HI titers were calculated by titrating 50 ml of antiserum treated with receptor-destroying enzyme and an equivalent amount of A/Vietnam/1203/2004 virus (four hemagglutinating doses) was added to each well. Wells were incubated at room temperature for 30 minutes and 50 ml of a 0.5% suspension of chicken red blood cells was added.", "Wells were incubated at room temperature for 30 minutes and 50 ml of a 0.5% suspension of chicken red blood cells was added. HI titers were calculated after 30 minutes as the reciprocal of the serum dilution that inhibited hemagglutination. Table S1 Hemagglutination inhibition (HI), microneutralization titer (NT), and LAI of sera from individual chickens immunized with different vaccines.", "Table S1 Hemagglutination inhibition (HI), microneutralization titer (NT), and LAI of sera from individual chickens immunized with different vaccines. Sera from immunized animals were obtained at week 5 or 7, a week before or after the final boost, and neutralization was assessed by HI, microneutralization (NT) and LAI (shown as IC 50 ). Individual animal serum of each group is shown and was analyzed as described in the Materials and Methods section.", "Individual animal serum of each group is shown and was analyzed as described in the Materials and Methods section. Figure S1 Characterization of needle-free (Agro-JetH) DNA immunization in chickens. To evaluate the distribution of fluid into superficial or deep layers of subcutaneous tissues after delivery by AgroJetH, 4 or 7 week old chickens were injected with a solution containing India ink with this needle-free device at various pressures, ranging from 45 to 55 mm Hg.", "To evaluate the distribution of fluid into superficial or deep layers of subcutaneous tissues after delivery by AgroJetH, 4 or 7 week old chickens were injected with a solution containing India ink with this needle-free device at various pressures, ranging from 45 to 55 mm Hg. Three sites (thigh, wing and breast) were used, and biopsies were taken for routine hematoxylin and eosin staining. Representative sections of thigh injections are shown from 7 week old chickens and were similar at 4 weeks (data not shown).", "Representative sections of thigh injections are shown from 7 week old chickens and were similar at 4 weeks (data not shown). While the 48 mm Hg pressure deposited the injectate into the dermis/subcutaneous region (left), the higher pressure injections, 52 and 58 mm Hg, deposited the injectate into the subcutaneous and muscle layers (middle, right). 48 mm Hg consistently provided an optimal pressure to deposit the injectate into the dermis and subcutaneous tissue and was chosen for all AgroJetH immunizations.", "48 mm Hg consistently provided an optimal pressure to deposit the injectate into the dermis and subcutaneous tissue and was chosen for all AgroJetH immunizations. Found at: .1371/journal.pone.0002432.s003 (10.74 MB DOC)" ]

[ "Background: Fulminant hepatitis (FH) is a serious threat to human life, accompanied by massive and rapid necroinflammation. Kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for FH. Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection.", "Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection. Clara cell 10 kDa (CC10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. However, its mechanisms of action and pathogenic roles in other disease are still unclear.", "However, its mechanisms of action and pathogenic roles in other disease are still unclear. In this study, we aimed to determine the role of CC10 in FH and the regulation of Fgl2 by CC10. Methods: A mouse FH model was established by peritoneal injection of MHV-3.", "Methods: A mouse FH model was established by peritoneal injection of MHV-3. The mice received CC10 protein through tail vein injection before viral infection. Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined.", "Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. The regulatory effect of CC10 on Fgl2 expression was investigated using THP-1 cells and mouse peritoneal macrophages in vitro. Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group.", "Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group. Meanwhile, the levels of ALT and AST in serum were significantly decreased and liver damage was reduced. Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein.", "Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein. In vitro, CC10 was found to significantly inhibit the expression of Fgl2 in IFN-γ-treated THP-1 cells and MHV-3-infected mouse peritoneal macrophages by western blot and real-time PCR. However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation.", "However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation. Microarray investigations suggested that HMG-box transcription factor 1 (HBP1) was significantly low in CC10-treated and IFN-γ-primed THP-1 cells. HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs).", "HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs). Conclusion:CC10 protects against MHV-3-induced FH via suppression of Fgl2 expression in macrophages. Such effects may be mediated by the transcription factor HBP1.", "Such effects may be mediated by the transcription factor HBP1. Text: Fulminant hepatitis (FH) is a serious life-threatening disease characterized by massive hepatocyte necrosis, severe liver damage, and high mortality. The underlying mechanisms and the pathogenesis of FH are not clear.", "The underlying mechanisms and the pathogenesis of FH are not clear. However, accumulating evidence suggests that, regardless of the pathogenesis of FH, the host's inflammatory responses contribute to liver microcirculatory disorders and injuries. Accordingly, It has been shown that immune cell activation and inflammatory cytokines play an important role in FH (1) .", "Accordingly, It has been shown that immune cell activation and inflammatory cytokines play an important role in FH (1) . In recent years, our laboratory has conducted extensive research on the pathogenesis of FH and found that immune cells play a key role in it. Kupffer cells, natural killer (NK) cells (2, 3) , cytotoxic T-lymphocytes (CTLs), and double negative T-cells (DNT) (4) (5) (6) in liver and the cytokines that are produced by these cells cause liver damage.", "Kupffer cells, natural killer (NK) cells (2, 3) , cytotoxic T-lymphocytes (CTLs), and double negative T-cells (DNT) (4) (5) (6) in liver and the cytokines that are produced by these cells cause liver damage. Prothrombinase Fgl2 belongs to the fibrinogen superfamily and is produced by activated macrophages or endothelial cells, transforming prothrombin directly into thrombin, so as to quickly initiate the process of coagulation. This promotes the conversion of fibrinogen into fibrin, resulting in thrombosis (7) (8) (9) (10) (11) (12) .", "This promotes the conversion of fibrinogen into fibrin, resulting in thrombosis (7) (8) (9) (10) (11) (12) . Our study found that Fgl2 was highly expressed in peripheral blood mononuclear cells (PBMCs) and in liver tissue of humans or mice with severe viral hepatitis, and was positively related to the severity of the disease (13, 14) . Gene therapy targeting Fgl2 silencing showed that the survival rate of fulminant hepatitis mice increased from 0 to 33.3% (15) .", "Gene therapy targeting Fgl2 silencing showed that the survival rate of fulminant hepatitis mice increased from 0 to 33.3% (15) . Thus far, the discovery and related research involving Fgl2 have provided new insights into the molecular mechanism of hepatocyte necrosis in FH. In view of the important role of Fgl2 in severe viral hepatitis, investigations concerning the regulation of Fgl2 will be beneficial in the search for new strategies for treatment of severe hepatitis.", "In view of the important role of Fgl2 in severe viral hepatitis, investigations concerning the regulation of Fgl2 will be beneficial in the search for new strategies for treatment of severe hepatitis. Clara cell 10 kDa protein (CC10), also considered to be uteroglobin, Clara cell secretory protein, is one of members of secretoglobin superfamily. Expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world (16) .", "Expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world (16) . CC10 has immunomodulatory and anti-inflammatory effects. Compared to wild-type mice, CC10-knockout mice exhibited excessive airway inflammation Abbreviations: FH, fulminant hepatitis; MHV-3, murine hepatitis virus strain 3; Fgl2, Fibrinogen-like protein 2; CC10, Clara cell 10 KDa protein; ALF, acute liver failure; PFU, plaque-forming units; PBS, phosphate-buffered saline; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCA, pro-coagulant activity; HRP, horseradish peroxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.", "Compared to wild-type mice, CC10-knockout mice exhibited excessive airway inflammation Abbreviations: FH, fulminant hepatitis; MHV-3, murine hepatitis virus strain 3; Fgl2, Fibrinogen-like protein 2; CC10, Clara cell 10 KDa protein; ALF, acute liver failure; PFU, plaque-forming units; PBS, phosphate-buffered saline; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCA, pro-coagulant activity; HRP, horseradish peroxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. caused by allergic reaction and bacterial and viral infections (17) . Reduced levels of CC10 are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis (18) (19) (20) (21) .", "Reduced levels of CC10 are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis (18) (19) (20) (21) . Previous studies and published articles show that CC10 protein can not only inhibit Th17 cell responses by inhibiting expression of related molecules of dendritic cells and cytokines in mice with allergic rhinitis, but also can inhibit chitosan-3 like protein 1 (22, 23) . Moreover, CC10 inhibits the expression of an important immune regulator, osteopontin (OPN), in models of allergic rhinitis (21) .", "Moreover, CC10 inhibits the expression of an important immune regulator, osteopontin (OPN), in models of allergic rhinitis (21) . In this study, we investigated the role of CC10 in hepatitis virus strain 3 (MHV-3)-induced FH in mice and explored whether CC10 protein could regulate Fgl2 in the disease process. Female BALB/cJ mice (Shanghai Shilaike Animal Seed Center, Shanghai, China), 6-8 weeks of age, with a body weight of 18.0-20.0 g, were kept in Tongji Hospital with food and water.", "Female BALB/cJ mice (Shanghai Shilaike Animal Seed Center, Shanghai, China), 6-8 weeks of age, with a body weight of 18.0-20.0 g, were kept in Tongji Hospital with food and water. Mice were divided into two groups: CC10 group (experimental group) and phosphate-buffered saline (PBS) group (control group). This study was carried out in accordance with the recommendations of the guidelines of the National Institutes of Health and the Animal Experiment Committee of Tongji hospital.", "This study was carried out in accordance with the recommendations of the guidelines of the National Institutes of Health and the Animal Experiment Committee of Tongji hospital. This study was reviewed and approved by the Animal Experiment Committee of Tongji hospital. The human monocyte cell line THP-1 was purchased from the Cell Institute of the Chinese Academy of Sciences (Shanghai, China).", "The human monocyte cell line THP-1 was purchased from the Cell Institute of the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from the Biology Treasure Center of Wuhan University, China. The Chinese hamster ovary (CHO) cell line was acquired from the typical culture preservation commission cell bank, the Chinese Academy of Sciences (Shanghai, China).", "The Chinese hamster ovary (CHO) cell line was acquired from the typical culture preservation commission cell bank, the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) and CHO cells were cultured in Dulbecco's modified Eagle's medium (DMEM), and THP-1 cells were maintained in RPMI 1,640 containing 10% heat inactivated fetal bovine serum (FBS, Gibco Life Technologies, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin and cultured at 37 • C, 50 mL/L CO 2 and 95% humidity. Peritoneal exudative macrophages (PEMs) were obtained from BALB/cJ mice.", "Peritoneal exudative macrophages (PEMs) were obtained from BALB/cJ mice. Cells were resuspended in RPMI 1,640 supplemented with 10% FBS at 1-2 × 10 6 cells/mL in a 6-well plate and incubated for 4 h. They were then washed with RPMI 1640 medium and non-adherent cells discarded. The adherent cells were macrophages and were incubated for a further 12 h. Peritoneal exudative macrophages (PEMs) were divided into two groups.", "The adherent cells were macrophages and were incubated for a further 12 h. Peritoneal exudative macrophages (PEMs) were divided into two groups. One group was supplemented with CC10 protein (150 ng/mL) and in the other group, PBS was added. After 2 h of stimulation, 1,000 plaque forming units (PFUs) of MHV-3 was added to the cells, which were then cultured for 4 h. Peritoneal exudative macrophages (PEMs) were harvested and lysed for real-time PCR and western blotting analysis.", "After 2 h of stimulation, 1,000 plaque forming units (PFUs) of MHV-3 was added to the cells, which were then cultured for 4 h. Peritoneal exudative macrophages (PEMs) were harvested and lysed for real-time PCR and western blotting analysis. Cell apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method with a TUNEL apoptosis detection kit (Roche, Switzerland). Briefly, 5 µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase K for 30 min at 37 • C. After stopping the proteinase K digestion reaction with PBS, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dUTP at a ratio of 2:29, respectively), for 2 h at 37 • C in an immunohistochemistry wet box.", "Briefly, 5 µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase K for 30 min at 37 • C. After stopping the proteinase K digestion reaction with PBS, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dUTP at a ratio of 2:29, respectively), for 2 h at 37 • C in an immunohistochemistry wet box. Following washing and blocking, each section was supplemented with reagent (converter-POD) to cover the tissues and incubated for 30 min at 37 • C in a wet box. Then, the liver tissue sections were washed with PBS, and colored with diaminobenzidine (DAB) subsequently.", "Then, the liver tissue sections were washed with PBS, and colored with diaminobenzidine (DAB) subsequently. Hepatocytes with nucleus stained brownish yellow were considered to be apoptotic cells. The expression of Fgl2 on THP-1 cells was measured by flow cytometry (BD FACS Canto II, USA).", "The expression of Fgl2 on THP-1 cells was measured by flow cytometry (BD FACS Canto II, USA). Briefly, cells (2 × 10 5 per tube) were incubated with Human TruStrain FcX (Fc Receptor Blocking solution, BioLegend, USA) for 10 min at room temperature and then incubated in the dark with mouse anti-Fgl2 antibody (1:100, Abnova,) or normal goat serum (an isotype control) at 4 • C for 40 min. Cells were washed with PBS and incubated in the dark with PE-conjugated goat anti-mouse IgG antibody (1:50, BioLegend, USA) at 4 • C for 30 min.", "Cells were washed with PBS and incubated in the dark with PE-conjugated goat anti-mouse IgG antibody (1:50, BioLegend, USA) at 4 • C for 30 min. Cells were then washed with PBS and resuspended in 300 µL PBS for study. Liver slices were fixed in 4% paraformaldehyde and then embedded in paraffin.", "Liver slices were fixed in 4% paraformaldehyde and then embedded in paraffin. Immunohistochemistry of liver tissues was performed using SP-9001 SPlink Detection Kits (Biotin-Streptavidin HRP Detection Systems) (ZSGB-BIO, Beijing, China) according to the manufacturer's instructions. For immunohistochemistry staining, the expression of Fgl2, fibrinogen, Fas and TNF-receptor 1 in mouse liver tissues was detected with polyclonal rabbit anti-mouse Fgl2 antibody (1:100, Proteintech, USA), polyclonal rabbit anti-mouse fibrinogen antibody (1:1,000, Abcam, EngLand), polyclonal rabbit antimouse Fas antibody (1:50, Abcam, EngLand), and polyclonal rabbit anti-mouse TNF-receptor 1 antibody (1:500, Abcam, EngLand), respectively.", "For immunohistochemistry staining, the expression of Fgl2, fibrinogen, Fas and TNF-receptor 1 in mouse liver tissues was detected with polyclonal rabbit anti-mouse Fgl2 antibody (1:100, Proteintech, USA), polyclonal rabbit anti-mouse fibrinogen antibody (1:1,000, Abcam, EngLand), polyclonal rabbit antimouse Fas antibody (1:50, Abcam, EngLand), and polyclonal rabbit anti-mouse TNF-receptor 1 antibody (1:500, Abcam, EngLand), respectively. After incubation with an horseradish peroxidase (HRP)-labeled goat IgG fraction to rabbit IgG Fc, the target protein was detected using a DAB kit (ZSGB-BIO, Beijing, China). The slides were then counterstained with hematoxylin and visualized under a microscope (Olympus, Tokyo, Japan).", "The slides were then counterstained with hematoxylin and visualized under a microscope (Olympus, Tokyo, Japan). Liver tissue and cells were homogenized in RIPA lysis buffer with phenyl methane sulfonyl fluoride (PMSF) protease inhibitor. Protein lysates were separated by SDS-PAGE, and western blotting was performed using a monoclonal mouse antihuman/mouse Fgl2 (1:750, Abnova), a monoclonal mouse antihuman HBP1 (1:100, Santa Cruz, USA), and a monoclonal rabbit anti-human/mouse β-actin (1:1,000, Cell Signaling Technology, USA).", "Protein lysates were separated by SDS-PAGE, and western blotting was performed using a monoclonal mouse antihuman/mouse Fgl2 (1:750, Abnova), a monoclonal mouse antihuman HBP1 (1:100, Santa Cruz, USA), and a monoclonal rabbit anti-human/mouse β-actin (1:1,000, Cell Signaling Technology, USA). Liver tissues were collected from MHV-3-infected BALB/cJ mice at 72 h, and total RNA was extracted using Trizol Reagent (Invitrogen, USA) and then reverse transcribed into cDNA by using ReverTra Ace qPCR RT kit (TOYOBO, Japan). The cDNA was then amplified by RT-PCR by using Dream Taq Green PCR Master Mix (2 ×) (Thermo Scientific, USA).", "The cDNA was then amplified by RT-PCR by using Dream Taq Green PCR Master Mix (2 ×) (Thermo Scientific, USA). Realtime quantitative PCR (qPCR) with SYBR Green Real-time PCR Master Mix (TOYOBO, Japan) was performed using a CFX96 real-time PCR detection system (Bio-Rad, USA) and mRNA levels were normalized with reference to those of the house keeping gene GAPDH. Primer sequences for qPCR amplification were as follows: mTNF-α forward, 5 ′ -TTT GAG ATC CAT GCC GTT GG-3 ′ ; mTNF-α reverse, 5 ′ -GCCA CCA CGC TCT TCT GT-3 ′ ; mIL-1β forward, 5 ′ -TGT AAT GAA AGA CGG CAC ACC-3 ′ ; mIL-1β reverse, 5 ′ -TCT TCT TTG GGT ATT GCT TGG-3 ′ .", "Primer sequences for qPCR amplification were as follows: mTNF-α forward, 5 ′ -TTT GAG ATC CAT GCC GTT GG-3 ′ ; mTNF-α reverse, 5 ′ -GCCA CCA CGC TCT TCT GT-3 ′ ; mIL-1β forward, 5 ′ -TGT AAT GAA AGA CGG CAC ACC-3 ′ ; mIL-1β reverse, 5 ′ -TCT TCT TTG GGT ATT GCT TGG-3 ′ . mFgl2 forward, 5 ′ -GCC AAA TGT GAG TCC CTG GAA-3 ′ ; mFgl2 reverse, 5 ′ -TTC CAC CCA AGA GCA CGT TTA AG-3 ′ ; hFgl2 forward 5 ′ -ACA GTT CAG GCT GGT GGT-3 ′ ; hFgl2 reverse, 5 ′ -GGC TTA AAG TGC TTG GGT-3 ′ ; HBP1 forward, 5 ′ -TGA AGC AGA AGC TGG GAGT-3 ′ ; HBP1 reverse,\n\nTHP-1 cells were treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma, USA) for 48 h to induce differentiation toward adherent macrophage-like cells as reported previously (24) . The CC10 group was supplemented with CC10 protein (150 ng/ml).", "The CC10 group was supplemented with CC10 protein (150 ng/ml). After 2 h of stimulation, IFN-γ (10 ng/ml) was added to these cells, which were then cultured for 12 h before they were collected for western blotting and real-time PCR studies. The Chinese hamster ovary (CHO) cells were cultured in 10 cm cell culture dishes with DMEM supplemented with 10% FBS until 80-90% confluence.", "The Chinese hamster ovary (CHO) cells were cultured in 10 cm cell culture dishes with DMEM supplemented with 10% FBS until 80-90% confluence. Next, 12 µg pcDNA3.1-hFgl2 (constructed in our lab) was mixed with 12 µg pcDNA3.1-hCC10 in serumfree DMEM. The mixture was then combined with Lipofectamine 2,000 (Invitrogen, USA) and mixed gently.", "The mixture was then combined with Lipofectamine 2,000 (Invitrogen, USA) and mixed gently. After incubation at 27 • C for 20 min, the solution was added to CHO cells and incubated at 37 • C in 5% CO 2 . Four to Six hour after transfection, the medium was removed and fresh medium containing 10% FBS was added.", "Four to Six hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, the cells were collected for co-immunoprecipitation analysis to evaluate the interaction of CC10 with Fgl2. Both HUVEC and THP-1 cells express fgl2.", "Both HUVEC and THP-1 cells express fgl2. However, in the transfection experiments, it is difficult to transfect the THP-1 cells with siRNA, so we use HUVEC instead of THP-1. Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in FIGURE 1 | CC10 protein increased survival rate and reduced liver damage in mice.", "Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in FIGURE 1 | CC10 protein increased survival rate and reduced liver damage in mice. (A) The survival rate of CC10 group is higher than the control group comprised of MHV-3-infected BALB/cJ mice treated with saline. CC10 protein (2 µg) or saline were injected into mice by tail vein.", "CC10 protein (2 µg) or saline were injected into mice by tail vein. BALB/cJ mice then received 100 PFU of MHV-3 intraperitoneally 24 h later to develop fulminant viral hepatitis. Then, CC10 protein (2 µg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later.", "Then, CC10 protein (2 µg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later. The survival rate was observed for 10 days (n = 24/group). Representative data from three independent experiments are shown. The survival curve was analyzed by using the Log-Rank Test.", "The survival curve was analyzed by using the Log-Rank Test. ***P < 0.001 compared with saline group. (B) Histopathology of liver tissues (H&E staining; original magnification, ×400, n = 5/group) at 72 h post-MHV-3 infection was evaluated in the two groups of MHV-3-infected BALB/cJ mice.", "(B) Histopathology of liver tissues (H&E staining; original magnification, ×400, n = 5/group) at 72 h post-MHV-3 infection was evaluated in the two groups of MHV-3-infected BALB/cJ mice. Livers were collected from saline-treated (a) and CC10-treated (b) BALB/cJ mice at 72 h after MHV-3 infection. Arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation.", "Arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation. (C) Effect of CC10 on serum ALT and AST levels (n = 6-8/group). Values represent means and standard error of three independent experiments performed in triplicate. **P < 0.01 compared with the saline group.", "**P < 0.01 compared with the saline group. six-well plates with DMEM supplemented with 10% FBS until 70-80% confluence. 50 pmol HBP1-siRNA was mixed with 125 µl serum-free DMEM. Two microliter Lipofectamine 2,000 was gently mixed with serum-free DMEM.", "Two microliter Lipofectamine 2,000 was gently mixed with serum-free DMEM. After incubation at 27 • C for 5 min, the solution was added to HUVECs and incubated at 37 • C. Four hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, cells were collected for real-time PCR and western blot analysis to evaluate the effects of HBP1 on Fgl2.", "At 48 h after transfection, cells were collected for real-time PCR and western blot analysis to evaluate the effects of HBP1 on Fgl2. At 24 h after transfection, the CC10 group was supplemented with the CC10 protein (150 ng/mL). After 4 h of stimulation, IFN-γ (10 ng/mL) was added to these cells.", "After 4 h of stimulation, IFN-γ (10 ng/mL) was added to these cells. These cells were then cultured for 24 h before they were harvested for real-time PCR studies to evaluate the effects of CC10 on Fgl2 by HBP1. Negative control was used as a control.", "Negative control was used as a control. To detect whether there was a potential interaction between CC10 protein and Fgl2, CHO cells were transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2 for 48 h. Cells transfected with empty plasmid pcDNA3.1 (mock) were used as negative controls for CC10 gene transfection. Immunoprecipitation and immunoblotting were performed by using Pierce Co-Immunoprecipitation Kit (Pierce, USA).", "Immunoprecipitation and immunoblotting were performed by using Pierce Co-Immunoprecipitation Kit (Pierce, USA). Total cell proteins were extracted as previously described (25) . The proteins were immunoprecipitated by mouse anti-human Fgl2 antibody (1:500, Abnova).", "The proteins were immunoprecipitated by mouse anti-human Fgl2 antibody (1:500, Abnova). For co-immunoprecipitation experiments, western blotting was performed using both rat anti-human uteroglobin/SCGB1A1 Antibody (1:750, R&D, USA) Frontiers in Immunology | and mouse anti-human Fgl2 antibody (1:500, Abnova). Control isotype rat IgG1 was used as a negative control for primary antibodies.", "Control isotype rat IgG1 was used as a negative control for primary antibodies. The human CC10 coding region gene, including a 389 bp sequence, was amplified from homogenized human turbinate tissue by RT-PCR. In this study, the sequences of PCR primers for CC10 were as follows: hCC10-forward, 5 ′ -CCC TCC ACC ATG AAA CTCG-3 ′ ; hCC10-reverse, 5 ′ -TGA GAT GCT TGT GGT TTA TTG AAG-3 ′ .", "In this study, the sequences of PCR primers for CC10 were as follows: hCC10-forward, 5 ′ -CCC TCC ACC ATG AAA CTCG-3 ′ ; hCC10-reverse, 5 ′ -TGA GAT GCT TGT GGT TTA TTG AAG-3 ′ . The PCR products were cloned into pEASY-T1 cloning vector (TransGEN, Beijing, China) and then subcloned into HindIII/XbaI site of pcDNA3.1 vector (Invitrogen, USA) to form eukaryotic expression plasmids pcDNA3.1-hCC10. Microarray analysis was used to screen changes in genome-wide gene expression patterns in THP-1 cells with or without CC10 protein.", "Microarray analysis was used to screen changes in genome-wide gene expression patterns in THP-1 cells with or without CC10 protein. The changes in over 47,000 human gene expression patterns were assessed using Affymetrix gene microarrays (Human Genome U133 Plus 2.0) (CapitalBio Co.,Ltd., Beijing, China). Three replicates were used for microarrays analysis.", "Three replicates were used for microarrays analysis. Data obtained from the experiments are expressed as means ± SEM. Survival curve comparisons were performed with the Log Rank test. Multiple group analyses for data were evaluated by one-way analyses of variance. Analyses of two group results were performed using Student's t-test to evaluate the statistical significance of differences.", "Analyses of two group results were performed using Student's t-test to evaluate the statistical significance of differences. Values of P < 0.05 indicated significance. To establish an animal model of mouse FH, MHV-3 was injected intraperitoneally to BALB/cJ mice (24 mice/group).", "To establish an animal model of mouse FH, MHV-3 was injected intraperitoneally to BALB/cJ mice (24 mice/group). To further study the role of CC10 in FH, recombinant mouse CC10 protein (2 µg/mouse) or saline was administrated into the tail vein 24 h prior to MHV-3 infection. The same dose of CC10 protein or saline was then administered 24 h later.", "The same dose of CC10 protein or saline was then administered 24 h later. The survival rate of the CC10 and saline groups was observed for 10 days. The results showed that mice in the two groups began to die at 48 h after injection of MHV-3 and exhibited symptoms of horripilation, slow activity, and reduced food consumption.", "The results showed that mice in the two groups began to die at 48 h after injection of MHV-3 and exhibited symptoms of horripilation, slow activity, and reduced food consumption. In the CC10 group 24 mice were alive on day 3 after infection, 4 mice alive on day 4, and 3 of 24 (12.5%) mice recovered from fulminant viral hepatitis. At the same time, in saline treated group, there were 5 mice alive on day 3, 1 mice alive on day 4 after infection, and no mice survived to day 5.", "At the same time, in saline treated group, there were 5 mice alive on day 3, 1 mice alive on day 4 after infection, and no mice survived to day 5. That is to say, the mice in the saline group died within 3 or 4 days. Three of 24 (12.5%) mice of the CC10 group recovered from fulminant viral hepatitis ( Figure 1A) .", "Three of 24 (12.5%) mice of the CC10 group recovered from fulminant viral hepatitis ( Figure 1A) . To better understand the mechanisms underlying the biological effects of the CC10 protein, liver function (ALT and AST levels in serum) and liver histology in mice of MHV-3-infected was performed. Liver tissues were harvested 72 h following MHV-3 infection, and liver histology was detected by H&E staining.", "Liver tissues were harvested 72 h following MHV-3 infection, and liver histology was detected by H&E staining. These results showed that there was substantial inflammatory cell infiltration and widespread necrosis of hepatocytes in the liver tissue of the saline group mice (Figure 1Ba ). There were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the CC10 group 72 h after MHV-3 infection (Figure 1Bb) .", "There were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the CC10 group 72 h after MHV-3 infection (Figure 1Bb) . Serum ALT and AST levels in mice were observed 72 h after MHV-3 infection. The results showed that serum ALT and AST levels in the saline group reached a peak 72 h after MHV-3 infection, but there was no significant increase in the CC10 group compared to the levels in the control group (P < 0.01, Figure 1C) .", "The results showed that serum ALT and AST levels in the saline group reached a peak 72 h after MHV-3 infection, but there was no significant increase in the CC10 group compared to the levels in the control group (P < 0.01, Figure 1C) . These results suggested that CC10 protein has a role in protection against MHV-3-induced liver injury in mice. To further elucidate the mechanisms of reduced liver injury following CC10 protein injection, we investigated the cytokines TNF-α and IL-1β expression.", "To further elucidate the mechanisms of reduced liver injury following CC10 protein injection, we investigated the cytokines TNF-α and IL-1β expression. Because these two cytokines play a crucial role in the liver damage of FH. They are characterized by an increase in apoptosis.", "They are characterized by an increase in apoptosis. Levels of TNF-α and IL-1β in liver tissues were markedly reduced in the CC10 group (as shown in Figure 2A) . Hepatic apoptosis (Figure 2B ) was significantly reduced in the CC10 group.", "Hepatic apoptosis (Figure 2B ) was significantly reduced in the CC10 group. We and collaborators have a long standing interest in studying the role of fgl2 in viral hepatitis. Fgl2 has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports.", "Fgl2 has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports. We have provided liver pathology figures and liver function for MHV-3 infected mice with a fgl2 gene knockout as shown in Supplementary Figure 1 . The data was comparable with previous reports from our center and collaborators.", "The data was comparable with previous reports from our center and collaborators. From this current study we shown that CC10 plays a protective role in liver damage.To study the related molecules of CC10 in MHV-3-induced FH mice, we evaluated whether there was crosstalk between Fgl2 and CC10. We found that the expression of Fgl2 in the liver of mice was reduced 72 h after MHV-3 infection and treatment with CC10 protein (Figures 3A,B) .", "We found that the expression of Fgl2 in the liver of mice was reduced 72 h after MHV-3 infection and treatment with CC10 protein (Figures 3A,B) . Furthermore, fibrin deposition, an indicator of liver injury associated with Fgl2 expression in FH, was also decreased in the livers of CC10-treated mice compared to that in controls (Figure 3C ). This indicates that CC10 treatment reduced liver injury after viral infection by inhibiting Fgl2 expression.", "This indicates that CC10 treatment reduced liver injury after viral infection by inhibiting Fgl2 expression. We examined the effect of increasing doses of CC10 protein (0, 50, 150, and 300 ng/mL) on IFN-γ-induced Fgl2 expression in THP-1 cells. CC10 treatment showed a 10.1% decrease in THP-1 cells compared to that in control after stimulation with 10 ng/mL IFN-γ for 12 h. CC10 protein inhibited Fgl2 expression between doses of 0 ng/mL and 300 ng/mL (Figure 4A ).", "CC10 treatment showed a 10.1% decrease in THP-1 cells compared to that in control after stimulation with 10 ng/mL IFN-γ for 12 h. CC10 protein inhibited Fgl2 expression between doses of 0 ng/mL and 300 ng/mL (Figure 4A ). In particular, 150 ng/mL CC10 protein had the strongest inhibitory effect on Fgl2 expression among the doses, and we chose this dose for the following experiments. We explored the effect of different time points of stimulation with a concentration of 150 ng/mL CC10 protein.", "We explored the effect of different time points of stimulation with a concentration of 150 ng/mL CC10 protein. After stimulation with CC10 protein for 6, 12, and 24 h compared to the PBS control, the strongest inhibitory effect on Fgl2 expression was noted at 12 h; hence, we chose this time point for the following studies ( Figure 4B ). An increasing number of studies suggest that macrophages are the primary source of Fgl2.", "An increasing number of studies suggest that macrophages are the primary source of Fgl2. In order to ascertain that CC10 has a direct effect on macrophages, we treated THP-1 cells with recombinant CC10 and assessed the expression of Fgl2. Unlike in controls, IFN-γ induced a significant increase in Fgl2 expression.", "Unlike in controls, IFN-γ induced a significant increase in Fgl2 expression. This effect was attenuated when cells were treated with CC10 protein (Figures 4C,D) , revealing that CC10 directly reduces the levels of Fgl2 in macrophages. To further explore the possibility that CC10 protein directly acts on macrophages, we infected murine PEMs with MHV-3 in the presence of recombinant CC10 and determined Fgl2 expression.", "To further explore the possibility that CC10 protein directly acts on macrophages, we infected murine PEMs with MHV-3 in the presence of recombinant CC10 and determined Fgl2 expression. Compared to levels in the controls, MHV-3infected macrophages exhibited a significant increase in Fgl2 production, and this effect was abolished by using CC10 protein (Figures 5A,B) , indicating that CC10 directly modulates Fgl2 production in macrophages. In order to determine genes that were downregulated after stimulation by CC10 protein, we used DNA microarray analysis to screen for differentially expressed genes.", "In order to determine genes that were downregulated after stimulation by CC10 protein, we used DNA microarray analysis to screen for differentially expressed genes. THP-1 cells were cultured and PMA was added to induce differentiation into macrophages. The production of Fgl2 was stimulated by IFNγ.", "The production of Fgl2 was stimulated by IFNγ. The experimental group was treated with CC10 protein for microarray detection of differentially expressed genes. The results showed that the most obviously downregulated genes were UBE2W, HECTD1, MIR612, ATRX, SOX4, HBP1, and Fgl2 (Supplementary Table 1) .", "The results showed that the most obviously downregulated genes were UBE2W, HECTD1, MIR612, ATRX, SOX4, HBP1, and Fgl2 (Supplementary Table 1) . And then these genes were tested by qPCR. However, UBE2W, HECTD1, MIR612, ATRX, and SOX4 was not differentially expressed by qPCR, while HBP1 and fgl2 were still down-regulated genes.", "However, UBE2W, HECTD1, MIR612, ATRX, and SOX4 was not differentially expressed by qPCR, while HBP1 and fgl2 were still down-regulated genes. DNA microarray analysis identified HBP1 as a down-regulated gene involved in the pathological processes of the regulation of CC10. Recently, very limited studies have explored the role of HBP1 in FH.", "Recently, very limited studies have explored the role of HBP1 in FH. Nevertheless, the mechanistic functions of HBP1 in FH remain largely unexplored. Therefore, we selected this gene for further study.", "Therefore, we selected this gene for further study. qPCR analysis confirmed that mRNA levels of HBP1 were significantly decreased in THP-1 cells after CC10\n\nprotein stimulation compared to that in the PBS control group (Figure 6A ). We knocked down HBP1 using HBP1-siRNA. Then, transfection of HBP1-SiRNA into HUVECs was detected by qPCR and western-blotting methods.", "Then, transfection of HBP1-SiRNA into HUVECs was detected by qPCR and western-blotting methods. As expected, HBP1 knockdown led to significantly decreased expression of HBP1 (Figures 6B,C) . Furthermore, HBP1 knockdown impaired expression of Fgl2 (Figure 6D ), suggesting that HBP1 was able to activate Fgl2. HBP1-SiRNA was used to transfect HUVECs.", "HBP1-SiRNA was used to transfect HUVECs. Then, IFN-γ was added to induce the expression of Fgl2 followed by stimulation with CC10 protein (150 ng/ml) after 2 h. Finally, we explored the expression of Fgl2 by qPCR. The results showed that HBP1-SiRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in HUVECs (Figure 7) .", "The results showed that HBP1-SiRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in HUVECs (Figure 7) . That is to say, CC10 could suppress Fgl2 expression in macrophages. Such an effect may be mediated by the transcription factor HBP1. It is well-known that CC10 protein can suppress the immune response.", "It is well-known that CC10 protein can suppress the immune response. In animal models of allergic diseases of the respiratory tract, most of evidences confirm this inhibition (26) . Its function in FH has not been investigated yet.", "Its function in FH has not been investigated yet. Here, we used a murine FH model established by MHV-3 infection to explore the effects of CC10 in this disease process. To determine the role of CC10 in the pathogenesis of FH, CC10 protein was injected into a mouse FH model established by MHV-3 infection.", "To determine the role of CC10 in the pathogenesis of FH, CC10 protein was injected into a mouse FH model established by MHV-3 infection. MHV-3-induced liver injury in CC10-treated mice occurred rarely and the areas of lesions were much fewer than those in saline-treated control mice. In summary, these results suggested that CC10 could reduce pathological liver damage in this FH model together with lower mortality rates followed by MHV-3 infection.", "In summary, these results suggested that CC10 could reduce pathological liver damage in this FH model together with lower mortality rates followed by MHV-3 infection. MHV-3 induced fulminant viral hepatitis progresses rapidly and infected mice die within 3-5 days. Previous studies suggested fgl2 played a vital role in this process with a 15-40% increase of survival when fgl2 was deleted (12, 15, 27, 28) .", "Previous studies suggested fgl2 played a vital role in this process with a 15-40% increase of survival when fgl2 was deleted (12, 15, 27, 28) . Multiple inflammatory factors or mediators including TNF-α and IFN-γ, IL-1β and C5aR have been demonstrated to promote FH progression with significant discrepancies between liver damage and survival rate (29) (30) (31) (32) , which is accordant with our observation that CC10 substantially alleviated liver injury though survival rate improved mildly. The survival rate based on hours may be more accurate to examine the effect of CC10 on FH.", "The survival rate based on hours may be more accurate to examine the effect of CC10 on FH. It is speculated that fgl2 can mediate lethality in MHV-3-induced FH. This is due to the fact that fgl2 induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity (15) .", "This is due to the fact that fgl2 induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity (15) . To determine whether the tissue necrosis was mediated by Fgl2 in CC10-treated mice following infection, Fgl2 expression was observed. Results suggested that the expression of Fgl2 was significantly increased in MHV-3-induced FH mice and CC10 treatment significantly reduced the production of Fgl2 in the infected liver and serum.", "Results suggested that the expression of Fgl2 was significantly increased in MHV-3-induced FH mice and CC10 treatment significantly reduced the production of Fgl2 in the infected liver and serum. In addition, decreased fibrinogen deposition was also observed in the livers of CC10-treated mice. Therefore, our research results strongly clarify that the lower mortality of CC10-treated mice after MHV-3 infection is due to the lower levels of Fgl2 and decreased fibrinogen deposition.", "Therefore, our research results strongly clarify that the lower mortality of CC10-treated mice after MHV-3 infection is due to the lower levels of Fgl2 and decreased fibrinogen deposition. Indeed, it has been reported that Fgl2 is expressed on macrophages, and the expression of Fgl2 is believed to be induced by IFN-γ and TNF-α (22) . Cultured THP-1 cells activated by IFN-γ or IL-2 have been demonstrated, with induction of Fgl2 expression and enhanced activation of human prothrombin (23) .", "Cultured THP-1 cells activated by IFN-γ or IL-2 have been demonstrated, with induction of Fgl2 expression and enhanced activation of human prothrombin (23) . Therefore, in this study, we explored this cell line to investigate the modulation of CC10 on Fgl2. Surprisingly, we found that CC10 directly inhibited IFN-γ-induced Fgl2 expression in THP-1 cells.", "Surprisingly, we found that CC10 directly inhibited IFN-γ-induced Fgl2 expression in THP-1 cells. As we know, IFN-γ has proved to be the main cytokine that leads to the development and progression of FH. Also, it was shown that IFN-γ might exert its own proinflammatory biological function through enhancing Fgl2 expression.", "Also, it was shown that IFN-γ might exert its own proinflammatory biological function through enhancing Fgl2 expression. Therefore, in our study, CC10 might counter the effect of IFN-γ in the setting of FH, which substantiates its role in FH. These results demonstrated that CC10 regulates the expression of Fgl2 in macrophages.", "These results demonstrated that CC10 regulates the expression of Fgl2 in macrophages. In the current study, we used co-immunoprecipitation to analyze binding between CC10 and Fgl2. In this study, we investigated possible protein-protein interactions between CC10 and Fgl2 in vitro. The Chinese hamster ovary (CHO) cells transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2.", "The Chinese hamster ovary (CHO) cells transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2. Cellular proteins were immunoprecipitated with anti-CC10 antibody or anti-Fgl2 antibody. Immunoblotting was performed with anti-Fgl2 and anti-CC10 antibodies.", "Immunoblotting was performed with anti-Fgl2 and anti-CC10 antibodies. Immunoprecipitation of protein extracts from pcDNA 3.1-CC10 and pcDNA3.1-Fgl2 co-transfected CHO cells with anti-Fgl2 or anti-CC10 antibody followed by western blotting with Fgl2 and CC10 antibodies indicated that CC10 did not co-immunoprecipitate with Fgl2, showing that there is no direct relationship between CC10 and Fgl2 (data not shown). The results showed that CC10 has no direct interaction with Fgl2.", "The results showed that CC10 has no direct interaction with Fgl2. From our previous study the gene of fgl2 contributed profoundly in MHV-3 induced fulminant hepatitis and is extensively expressed in macrophages and endothelium (12, 33) . Our microarray indicated a CC10 down-regulated fgl2 expression and this is further confirmed by qPCR and Western blotting in vivo (peritoneal macrophages) and in vitro (THP-1, macrophage cell line).", "Our microarray indicated a CC10 down-regulated fgl2 expression and this is further confirmed by qPCR and Western blotting in vivo (peritoneal macrophages) and in vitro (THP-1, macrophage cell line). Therefore, it is reasonable to focus on macrophages to display the effect of CC10 on fgl2 expression and eventually mice survival. We entirely agree there may be other possibilities for a protective effect of CC10 to contribute to the disease process.", "We entirely agree there may be other possibilities for a protective effect of CC10 to contribute to the disease process. This is worth further studies. The potential receptor of CC10 has not been revealed yet. Our previous study have demonstrated that CC10 have effect of dendritic cells in allergic rhinitis (34) .", "Our previous study have demonstrated that CC10 have effect of dendritic cells in allergic rhinitis (34) . In this research, we evaluated the effect of CC10 on macrophages functions and found Fgl2 was substantially down-regulated upon CC10 treatment, therefore, we speculate that potential CC10 receptor may be also expressed on macrophages. The potential target of CC10 on other immune cells cannot be excluded.", "The potential target of CC10 on other immune cells cannot be excluded. DNA microarray analysis is one of the most powerful approaches for the potential identification of unexpected genes involved in pathogenic processes. By using this approach, HMGbox transcription factor 1 (HBP1) was found to be one of the most downregulated genes after CC10 treatment of THP-1 cells.", "By using this approach, HMGbox transcription factor 1 (HBP1) was found to be one of the most downregulated genes after CC10 treatment of THP-1 cells. HBP1 is a well-described transcriptional repressor that modulates expression of genes involved in cell cycle progression. In a recent study, it was found that HBP1 is a direct target of miR-21 and confirmed that HBP1 modulates the inhibitory function of miR-21-ASO in hepatosteatosis and carcinogenesis simultaneously (23) .", "In a recent study, it was found that HBP1 is a direct target of miR-21 and confirmed that HBP1 modulates the inhibitory function of miR-21-ASO in hepatosteatosis and carcinogenesis simultaneously (23) . HBP1 is an endogenous inhibitor of the Wnt signaling pathway in both normal and cancer cells. The tumor suppressor role of HBP1 has been reported in some malignancies, such as oral cancer and glioma (35) .", "The tumor suppressor role of HBP1 has been reported in some malignancies, such as oral cancer and glioma (35) . However, an association between HBP1 and Fgl2 has not been investigated yet. The current study clearly demonstrated that CC10 protects against MHV-3 induced FH via suppression of Fgl2 expression. Such effects might be mediated by HBP1.", "Such effects might be mediated by HBP1. However, the functional status of HBP1 in the CC10 pathway requires further research, and such studies are conducting in our laboratory. In conclusion, we demonstrated that CC10 could limit the immunopathological damage in MHV-3-induced FH mice.", "In conclusion, we demonstrated that CC10 could limit the immunopathological damage in MHV-3-induced FH mice. Our results suggest that enhancing CC10 expression by an immunotherapeutic approach might be an effective treatment for FH. HY performed all the described experiments and wrote the manuscript. YL assisted with some experiments, analyzed experimental results, and edited the manuscript.", "YL assisted with some experiments, analyzed experimental results, and edited the manuscript. HW analyzed experimental results. XW reviewed and edited the manuscript. JH, WY, DX, XL, GS, and QN provided experimental help and design." ]

[ "Background: Fulminant hepatitis (FH) is a serious threat to human life, accompanied by massive and rapid necroinflammation. Kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for FH. Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection.", "Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection. Clara cell 10 kDa (CC10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. However, its mechanisms of action and pathogenic roles in other disease are still unclear.", "However, its mechanisms of action and pathogenic roles in other disease are still unclear. In this study, we aimed to determine the role of CC10 in FH and the regulation of Fgl2 by CC10. Methods: A mouse FH model was established by peritoneal injection of MHV-3.", "Methods: A mouse FH model was established by peritoneal injection of MHV-3. The mice received CC10 protein through tail vein injection before viral infection. Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined.", "Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. The regulatory effect of CC10 on Fgl2 expression was investigated using THP-1 cells and mouse peritoneal macrophages in vitro. Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group.", "Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group. Meanwhile, the levels of ALT and AST in serum were significantly decreased and liver damage was reduced. Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein.", "Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein. In vitro, CC10 was found to significantly inhibit the expression of Fgl2 in IFN-γ-treated THP-1 cells and MHV-3-infected mouse peritoneal macrophages by western blot and real-time PCR. However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation.", "However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation. Microarray investigations suggested that HMG-box transcription factor 1 (HBP1) was significantly low in CC10-treated and IFN-γ-primed THP-1 cells. HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs).", "HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs). Conclusion:CC10 protects against MHV-3-induced FH via suppression of Fgl2 expression in macrophages. Such effects may be mediated by the transcription factor HBP1.", "Such effects may be mediated by the transcription factor HBP1. Text: Fulminant hepatitis (FH) is a serious life-threatening disease characterized by massive hepatocyte necrosis, severe liver damage, and high mortality. The underlying mechanisms and the pathogenesis of FH are not clear.", "The underlying mechanisms and the pathogenesis of FH are not clear. However, accumulating evidence suggests that, regardless of the pathogenesis of FH, the host's inflammatory responses contribute to liver microcirculatory disorders and injuries. Accordingly, It has been shown that immune cell activation and inflammatory cytokines play an important role in FH (1) .", "Accordingly, It has been shown that immune cell activation and inflammatory cytokines play an important role in FH (1) . In recent years, our laboratory has conducted extensive research on the pathogenesis of FH and found that immune cells play a key role in it. Kupffer cells, natural killer (NK) cells (2, 3) , cytotoxic T-lymphocytes (CTLs), and double negative T-cells (DNT) (4) (5) (6) in liver and the cytokines that are produced by these cells cause liver damage.", "Kupffer cells, natural killer (NK) cells (2, 3) , cytotoxic T-lymphocytes (CTLs), and double negative T-cells (DNT) (4) (5) (6) in liver and the cytokines that are produced by these cells cause liver damage. Prothrombinase Fgl2 belongs to the fibrinogen superfamily and is produced by activated macrophages or endothelial cells, transforming prothrombin directly into thrombin, so as to quickly initiate the process of coagulation. This promotes the conversion of fibrinogen into fibrin, resulting in thrombosis (7) (8) (9) (10) (11) (12) .", "This promotes the conversion of fibrinogen into fibrin, resulting in thrombosis (7) (8) (9) (10) (11) (12) . Our study found that Fgl2 was highly expressed in peripheral blood mononuclear cells (PBMCs) and in liver tissue of humans or mice with severe viral hepatitis, and was positively related to the severity of the disease (13, 14) . Gene therapy targeting Fgl2 silencing showed that the survival rate of fulminant hepatitis mice increased from 0 to 33.3% (15) .", "Gene therapy targeting Fgl2 silencing showed that the survival rate of fulminant hepatitis mice increased from 0 to 33.3% (15) . Thus far, the discovery and related research involving Fgl2 have provided new insights into the molecular mechanism of hepatocyte necrosis in FH. In view of the important role of Fgl2 in severe viral hepatitis, investigations concerning the regulation of Fgl2 will be beneficial in the search for new strategies for treatment of severe hepatitis.", "In view of the important role of Fgl2 in severe viral hepatitis, investigations concerning the regulation of Fgl2 will be beneficial in the search for new strategies for treatment of severe hepatitis. Clara cell 10 kDa protein (CC10), also considered to be uteroglobin, Clara cell secretory protein, is one of members of secretoglobin superfamily. Expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world (16) .", "Expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world (16) . CC10 has immunomodulatory and anti-inflammatory effects. Compared to wild-type mice, CC10-knockout mice exhibited excessive airway inflammation Abbreviations: FH, fulminant hepatitis; MHV-3, murine hepatitis virus strain 3; Fgl2, Fibrinogen-like protein 2; CC10, Clara cell 10 KDa protein; ALF, acute liver failure; PFU, plaque-forming units; PBS, phosphate-buffered saline; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCA, pro-coagulant activity; HRP, horseradish peroxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.", "Compared to wild-type mice, CC10-knockout mice exhibited excessive airway inflammation Abbreviations: FH, fulminant hepatitis; MHV-3, murine hepatitis virus strain 3; Fgl2, Fibrinogen-like protein 2; CC10, Clara cell 10 KDa protein; ALF, acute liver failure; PFU, plaque-forming units; PBS, phosphate-buffered saline; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCA, pro-coagulant activity; HRP, horseradish peroxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. caused by allergic reaction and bacterial and viral infections (17) . Reduced levels of CC10 are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis (18) (19) (20) (21) .", "Reduced levels of CC10 are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis (18) (19) (20) (21) . Previous studies and published articles show that CC10 protein can not only inhibit Th17 cell responses by inhibiting expression of related molecules of dendritic cells and cytokines in mice with allergic rhinitis, but also can inhibit chitosan-3 like protein 1 (22, 23) . Moreover, CC10 inhibits the expression of an important immune regulator, osteopontin (OPN), in models of allergic rhinitis (21) .", "Moreover, CC10 inhibits the expression of an important immune regulator, osteopontin (OPN), in models of allergic rhinitis (21) . In this study, we investigated the role of CC10 in hepatitis virus strain 3 (MHV-3)-induced FH in mice and explored whether CC10 protein could regulate Fgl2 in the disease process. Female BALB/cJ mice (Shanghai Shilaike Animal Seed Center, Shanghai, China), 6-8 weeks of age, with a body weight of 18.0-20.0 g, were kept in Tongji Hospital with food and water.", "Female BALB/cJ mice (Shanghai Shilaike Animal Seed Center, Shanghai, China), 6-8 weeks of age, with a body weight of 18.0-20.0 g, were kept in Tongji Hospital with food and water. Mice were divided into two groups: CC10 group (experimental group) and phosphate-buffered saline (PBS) group (control group). This study was carried out in accordance with the recommendations of the guidelines of the National Institutes of Health and the Animal Experiment Committee of Tongji hospital.", "This study was carried out in accordance with the recommendations of the guidelines of the National Institutes of Health and the Animal Experiment Committee of Tongji hospital. This study was reviewed and approved by the Animal Experiment Committee of Tongji hospital. The human monocyte cell line THP-1 was purchased from the Cell Institute of the Chinese Academy of Sciences (Shanghai, China).", "The human monocyte cell line THP-1 was purchased from the Cell Institute of the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from the Biology Treasure Center of Wuhan University, China. The Chinese hamster ovary (CHO) cell line was acquired from the typical culture preservation commission cell bank, the Chinese Academy of Sciences (Shanghai, China).", "The Chinese hamster ovary (CHO) cell line was acquired from the typical culture preservation commission cell bank, the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) and CHO cells were cultured in Dulbecco's modified Eagle's medium (DMEM), and THP-1 cells were maintained in RPMI 1,640 containing 10% heat inactivated fetal bovine serum (FBS, Gibco Life Technologies, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin and cultured at 37 • C, 50 mL/L CO 2 and 95% humidity. Peritoneal exudative macrophages (PEMs) were obtained from BALB/cJ mice.", "Peritoneal exudative macrophages (PEMs) were obtained from BALB/cJ mice. Cells were resuspended in RPMI 1,640 supplemented with 10% FBS at 1-2 × 10 6 cells/mL in a 6-well plate and incubated for 4 h. They were then washed with RPMI 1640 medium and non-adherent cells discarded. The adherent cells were macrophages and were incubated for a further 12 h. Peritoneal exudative macrophages (PEMs) were divided into two groups.", "The adherent cells were macrophages and were incubated for a further 12 h. Peritoneal exudative macrophages (PEMs) were divided into two groups. One group was supplemented with CC10 protein (150 ng/mL) and in the other group, PBS was added. After 2 h of stimulation, 1,000 plaque forming units (PFUs) of MHV-3 was added to the cells, which were then cultured for 4 h. Peritoneal exudative macrophages (PEMs) were harvested and lysed for real-time PCR and western blotting analysis.", "After 2 h of stimulation, 1,000 plaque forming units (PFUs) of MHV-3 was added to the cells, which were then cultured for 4 h. Peritoneal exudative macrophages (PEMs) were harvested and lysed for real-time PCR and western blotting analysis. Cell apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method with a TUNEL apoptosis detection kit (Roche, Switzerland). Briefly, 5 µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase K for 30 min at 37 • C. After stopping the proteinase K digestion reaction with PBS, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dUTP at a ratio of 2:29, respectively), for 2 h at 37 • C in an immunohistochemistry wet box.", "Briefly, 5 µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase K for 30 min at 37 • C. After stopping the proteinase K digestion reaction with PBS, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dUTP at a ratio of 2:29, respectively), for 2 h at 37 • C in an immunohistochemistry wet box. Following washing and blocking, each section was supplemented with reagent (converter-POD) to cover the tissues and incubated for 30 min at 37 • C in a wet box. Then, the liver tissue sections were washed with PBS, and colored with diaminobenzidine (DAB) subsequently.", "Then, the liver tissue sections were washed with PBS, and colored with diaminobenzidine (DAB) subsequently. Hepatocytes with nucleus stained brownish yellow were considered to be apoptotic cells. The expression of Fgl2 on THP-1 cells was measured by flow cytometry (BD FACS Canto II, USA).", "The expression of Fgl2 on THP-1 cells was measured by flow cytometry (BD FACS Canto II, USA). Briefly, cells (2 × 10 5 per tube) were incubated with Human TruStrain FcX (Fc Receptor Blocking solution, BioLegend, USA) for 10 min at room temperature and then incubated in the dark with mouse anti-Fgl2 antibody (1:100, Abnova,) or normal goat serum (an isotype control) at 4 • C for 40 min. Cells were washed with PBS and incubated in the dark with PE-conjugated goat anti-mouse IgG antibody (1:50, BioLegend, USA) at 4 • C for 30 min.", "Cells were washed with PBS and incubated in the dark with PE-conjugated goat anti-mouse IgG antibody (1:50, BioLegend, USA) at 4 • C for 30 min. Cells were then washed with PBS and resuspended in 300 µL PBS for study. Liver slices were fixed in 4% paraformaldehyde and then embedded in paraffin.", "Liver slices were fixed in 4% paraformaldehyde and then embedded in paraffin. Immunohistochemistry of liver tissues was performed using SP-9001 SPlink Detection Kits (Biotin-Streptavidin HRP Detection Systems) (ZSGB-BIO, Beijing, China) according to the manufacturer's instructions. For immunohistochemistry staining, the expression of Fgl2, fibrinogen, Fas and TNF-receptor 1 in mouse liver tissues was detected with polyclonal rabbit anti-mouse Fgl2 antibody (1:100, Proteintech, USA), polyclonal rabbit anti-mouse fibrinogen antibody (1:1,000, Abcam, EngLand), polyclonal rabbit antimouse Fas antibody (1:50, Abcam, EngLand), and polyclonal rabbit anti-mouse TNF-receptor 1 antibody (1:500, Abcam, EngLand), respectively.", "For immunohistochemistry staining, the expression of Fgl2, fibrinogen, Fas and TNF-receptor 1 in mouse liver tissues was detected with polyclonal rabbit anti-mouse Fgl2 antibody (1:100, Proteintech, USA), polyclonal rabbit anti-mouse fibrinogen antibody (1:1,000, Abcam, EngLand), polyclonal rabbit antimouse Fas antibody (1:50, Abcam, EngLand), and polyclonal rabbit anti-mouse TNF-receptor 1 antibody (1:500, Abcam, EngLand), respectively. After incubation with an horseradish peroxidase (HRP)-labeled goat IgG fraction to rabbit IgG Fc, the target protein was detected using a DAB kit (ZSGB-BIO, Beijing, China). The slides were then counterstained with hematoxylin and visualized under a microscope (Olympus, Tokyo, Japan).", "The slides were then counterstained with hematoxylin and visualized under a microscope (Olympus, Tokyo, Japan). Liver tissue and cells were homogenized in RIPA lysis buffer with phenyl methane sulfonyl fluoride (PMSF) protease inhibitor. Protein lysates were separated by SDS-PAGE, and western blotting was performed using a monoclonal mouse antihuman/mouse Fgl2 (1:750, Abnova), a monoclonal mouse antihuman HBP1 (1:100, Santa Cruz, USA), and a monoclonal rabbit anti-human/mouse β-actin (1:1,000, Cell Signaling Technology, USA).", "Protein lysates were separated by SDS-PAGE, and western blotting was performed using a monoclonal mouse antihuman/mouse Fgl2 (1:750, Abnova), a monoclonal mouse antihuman HBP1 (1:100, Santa Cruz, USA), and a monoclonal rabbit anti-human/mouse β-actin (1:1,000, Cell Signaling Technology, USA). Liver tissues were collected from MHV-3-infected BALB/cJ mice at 72 h, and total RNA was extracted using Trizol Reagent (Invitrogen, USA) and then reverse transcribed into cDNA by using ReverTra Ace qPCR RT kit (TOYOBO, Japan). The cDNA was then amplified by RT-PCR by using Dream Taq Green PCR Master Mix (2 ×) (Thermo Scientific, USA).", "The cDNA was then amplified by RT-PCR by using Dream Taq Green PCR Master Mix (2 ×) (Thermo Scientific, USA). Realtime quantitative PCR (qPCR) with SYBR Green Real-time PCR Master Mix (TOYOBO, Japan) was performed using a CFX96 real-time PCR detection system (Bio-Rad, USA) and mRNA levels were normalized with reference to those of the house keeping gene GAPDH. Primer sequences for qPCR amplification were as follows: mTNF-α forward, 5 ′ -TTT GAG ATC CAT GCC GTT GG-3 ′ ; mTNF-α reverse, 5 ′ -GCCA CCA CGC TCT TCT GT-3 ′ ; mIL-1β forward, 5 ′ -TGT AAT GAA AGA CGG CAC ACC-3 ′ ; mIL-1β reverse, 5 ′ -TCT TCT TTG GGT ATT GCT TGG-3 ′ .", "Primer sequences for qPCR amplification were as follows: mTNF-α forward, 5 ′ -TTT GAG ATC CAT GCC GTT GG-3 ′ ; mTNF-α reverse, 5 ′ -GCCA CCA CGC TCT TCT GT-3 ′ ; mIL-1β forward, 5 ′ -TGT AAT GAA AGA CGG CAC ACC-3 ′ ; mIL-1β reverse, 5 ′ -TCT TCT TTG GGT ATT GCT TGG-3 ′ . mFgl2 forward, 5 ′ -GCC AAA TGT GAG TCC CTG GAA-3 ′ ; mFgl2 reverse, 5 ′ -TTC CAC CCA AGA GCA CGT TTA AG-3 ′ ; hFgl2 forward 5 ′ -ACA GTT CAG GCT GGT GGT-3 ′ ; hFgl2 reverse, 5 ′ -GGC TTA AAG TGC TTG GGT-3 ′ ; HBP1 forward, 5 ′ -TGA AGC AGA AGC TGG GAGT-3 ′ ; HBP1 reverse,\n\nTHP-1 cells were treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma, USA) for 48 h to induce differentiation toward adherent macrophage-like cells as reported previously (24) . The CC10 group was supplemented with CC10 protein (150 ng/ml).", "The CC10 group was supplemented with CC10 protein (150 ng/ml). After 2 h of stimulation, IFN-γ (10 ng/ml) was added to these cells, which were then cultured for 12 h before they were collected for western blotting and real-time PCR studies. The Chinese hamster ovary (CHO) cells were cultured in 10 cm cell culture dishes with DMEM supplemented with 10% FBS until 80-90% confluence.", "The Chinese hamster ovary (CHO) cells were cultured in 10 cm cell culture dishes with DMEM supplemented with 10% FBS until 80-90% confluence. Next, 12 µg pcDNA3.1-hFgl2 (constructed in our lab) was mixed with 12 µg pcDNA3.1-hCC10 in serumfree DMEM. The mixture was then combined with Lipofectamine 2,000 (Invitrogen, USA) and mixed gently.", "The mixture was then combined with Lipofectamine 2,000 (Invitrogen, USA) and mixed gently. After incubation at 27 • C for 20 min, the solution was added to CHO cells and incubated at 37 • C in 5% CO 2 . Four to Six hour after transfection, the medium was removed and fresh medium containing 10% FBS was added.", "Four to Six hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, the cells were collected for co-immunoprecipitation analysis to evaluate the interaction of CC10 with Fgl2. Both HUVEC and THP-1 cells express fgl2.", "Both HUVEC and THP-1 cells express fgl2. However, in the transfection experiments, it is difficult to transfect the THP-1 cells with siRNA, so we use HUVEC instead of THP-1. Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in FIGURE 1 | CC10 protein increased survival rate and reduced liver damage in mice.", "Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in FIGURE 1 | CC10 protein increased survival rate and reduced liver damage in mice. (A) The survival rate of CC10 group is higher than the control group comprised of MHV-3-infected BALB/cJ mice treated with saline. CC10 protein (2 µg) or saline were injected into mice by tail vein.", "CC10 protein (2 µg) or saline were injected into mice by tail vein. BALB/cJ mice then received 100 PFU of MHV-3 intraperitoneally 24 h later to develop fulminant viral hepatitis. Then, CC10 protein (2 µg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later.", "Then, CC10 protein (2 µg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later. The survival rate was observed for 10 days (n = 24/group). Representative data from three independent experiments are shown. The survival curve was analyzed by using the Log-Rank Test.", "The survival curve was analyzed by using the Log-Rank Test. ***P < 0.001 compared with saline group. (B) Histopathology of liver tissues (H&E staining; original magnification, ×400, n = 5/group) at 72 h post-MHV-3 infection was evaluated in the two groups of MHV-3-infected BALB/cJ mice.", "(B) Histopathology of liver tissues (H&E staining; original magnification, ×400, n = 5/group) at 72 h post-MHV-3 infection was evaluated in the two groups of MHV-3-infected BALB/cJ mice. Livers were collected from saline-treated (a) and CC10-treated (b) BALB/cJ mice at 72 h after MHV-3 infection. Arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation.", "Arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation. (C) Effect of CC10 on serum ALT and AST levels (n = 6-8/group). Values represent means and standard error of three independent experiments performed in triplicate. **P < 0.01 compared with the saline group.", "**P < 0.01 compared with the saline group. six-well plates with DMEM supplemented with 10% FBS until 70-80% confluence. 50 pmol HBP1-siRNA was mixed with 125 µl serum-free DMEM. Two microliter Lipofectamine 2,000 was gently mixed with serum-free DMEM.", "Two microliter Lipofectamine 2,000 was gently mixed with serum-free DMEM. After incubation at 27 • C for 5 min, the solution was added to HUVECs and incubated at 37 • C. Four hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, cells were collected for real-time PCR and western blot analysis to evaluate the effects of HBP1 on Fgl2.", "At 48 h after transfection, cells were collected for real-time PCR and western blot analysis to evaluate the effects of HBP1 on Fgl2. At 24 h after transfection, the CC10 group was supplemented with the CC10 protein (150 ng/mL). After 4 h of stimulation, IFN-γ (10 ng/mL) was added to these cells.", "After 4 h of stimulation, IFN-γ (10 ng/mL) was added to these cells. These cells were then cultured for 24 h before they were harvested for real-time PCR studies to evaluate the effects of CC10 on Fgl2 by HBP1. Negative control was used as a control.", "Negative control was used as a control. To detect whether there was a potential interaction between CC10 protein and Fgl2, CHO cells were transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2 for 48 h. Cells transfected with empty plasmid pcDNA3.1 (mock) were used as negative controls for CC10 gene transfection. Immunoprecipitation and immunoblotting were performed by using Pierce Co-Immunoprecipitation Kit (Pierce, USA).", "Immunoprecipitation and immunoblotting were performed by using Pierce Co-Immunoprecipitation Kit (Pierce, USA). Total cell proteins were extracted as previously described (25) . The proteins were immunoprecipitated by mouse anti-human Fgl2 antibody (1:500, Abnova).", "The proteins were immunoprecipitated by mouse anti-human Fgl2 antibody (1:500, Abnova). For co-immunoprecipitation experiments, western blotting was performed using both rat anti-human uteroglobin/SCGB1A1 Antibody (1:750, R&D, USA) Frontiers in Immunology | and mouse anti-human Fgl2 antibody (1:500, Abnova). Control isotype rat IgG1 was used as a negative control for primary antibodies.", "Control isotype rat IgG1 was used as a negative control for primary antibodies. The human CC10 coding region gene, including a 389 bp sequence, was amplified from homogenized human turbinate tissue by RT-PCR. In this study, the sequences of PCR primers for CC10 were as follows: hCC10-forward, 5 ′ -CCC TCC ACC ATG AAA CTCG-3 ′ ; hCC10-reverse, 5 ′ -TGA GAT GCT TGT GGT TTA TTG AAG-3 ′ .", "In this study, the sequences of PCR primers for CC10 were as follows: hCC10-forward, 5 ′ -CCC TCC ACC ATG AAA CTCG-3 ′ ; hCC10-reverse, 5 ′ -TGA GAT GCT TGT GGT TTA TTG AAG-3 ′ . The PCR products were cloned into pEASY-T1 cloning vector (TransGEN, Beijing, China) and then subcloned into HindIII/XbaI site of pcDNA3.1 vector (Invitrogen, USA) to form eukaryotic expression plasmids pcDNA3.1-hCC10. Microarray analysis was used to screen changes in genome-wide gene expression patterns in THP-1 cells with or without CC10 protein.", "Microarray analysis was used to screen changes in genome-wide gene expression patterns in THP-1 cells with or without CC10 protein. The changes in over 47,000 human gene expression patterns were assessed using Affymetrix gene microarrays (Human Genome U133 Plus 2.0) (CapitalBio Co.,Ltd., Beijing, China). Three replicates were used for microarrays analysis.", "Three replicates were used for microarrays analysis. Data obtained from the experiments are expressed as means ± SEM. Survival curve comparisons were performed with the Log Rank test. Multiple group analyses for data were evaluated by one-way analyses of variance. Analyses of two group results were performed using Student's t-test to evaluate the statistical significance of differences.", "Analyses of two group results were performed using Student's t-test to evaluate the statistical significance of differences. Values of P < 0.05 indicated significance. To establish an animal model of mouse FH, MHV-3 was injected intraperitoneally to BALB/cJ mice (24 mice/group).", "To establish an animal model of mouse FH, MHV-3 was injected intraperitoneally to BALB/cJ mice (24 mice/group). To further study the role of CC10 in FH, recombinant mouse CC10 protein (2 µg/mouse) or saline was administrated into the tail vein 24 h prior to MHV-3 infection. The same dose of CC10 protein or saline was then administered 24 h later.", "The same dose of CC10 protein or saline was then administered 24 h later. The survival rate of the CC10 and saline groups was observed for 10 days. The results showed that mice in the two groups began to die at 48 h after injection of MHV-3 and exhibited symptoms of horripilation, slow activity, and reduced food consumption.", "The results showed that mice in the two groups began to die at 48 h after injection of MHV-3 and exhibited symptoms of horripilation, slow activity, and reduced food consumption. In the CC10 group 24 mice were alive on day 3 after infection, 4 mice alive on day 4, and 3 of 24 (12.5%) mice recovered from fulminant viral hepatitis. At the same time, in saline treated group, there were 5 mice alive on day 3, 1 mice alive on day 4 after infection, and no mice survived to day 5.", "At the same time, in saline treated group, there were 5 mice alive on day 3, 1 mice alive on day 4 after infection, and no mice survived to day 5. That is to say, the mice in the saline group died within 3 or 4 days. Three of 24 (12.5%) mice of the CC10 group recovered from fulminant viral hepatitis ( Figure 1A) .", "Three of 24 (12.5%) mice of the CC10 group recovered from fulminant viral hepatitis ( Figure 1A) . To better understand the mechanisms underlying the biological effects of the CC10 protein, liver function (ALT and AST levels in serum) and liver histology in mice of MHV-3-infected was performed. Liver tissues were harvested 72 h following MHV-3 infection, and liver histology was detected by H&E staining.", "Liver tissues were harvested 72 h following MHV-3 infection, and liver histology was detected by H&E staining. These results showed that there was substantial inflammatory cell infiltration and widespread necrosis of hepatocytes in the liver tissue of the saline group mice (Figure 1Ba ). There were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the CC10 group 72 h after MHV-3 infection (Figure 1Bb) .", "There were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the CC10 group 72 h after MHV-3 infection (Figure 1Bb) . Serum ALT and AST levels in mice were observed 72 h after MHV-3 infection. The results showed that serum ALT and AST levels in the saline group reached a peak 72 h after MHV-3 infection, but there was no significant increase in the CC10 group compared to the levels in the control group (P < 0.01, Figure 1C) .", "The results showed that serum ALT and AST levels in the saline group reached a peak 72 h after MHV-3 infection, but there was no significant increase in the CC10 group compared to the levels in the control group (P < 0.01, Figure 1C) . These results suggested that CC10 protein has a role in protection against MHV-3-induced liver injury in mice. To further elucidate the mechanisms of reduced liver injury following CC10 protein injection, we investigated the cytokines TNF-α and IL-1β expression.", "To further elucidate the mechanisms of reduced liver injury following CC10 protein injection, we investigated the cytokines TNF-α and IL-1β expression. Because these two cytokines play a crucial role in the liver damage of FH. They are characterized by an increase in apoptosis.", "They are characterized by an increase in apoptosis. Levels of TNF-α and IL-1β in liver tissues were markedly reduced in the CC10 group (as shown in Figure 2A) . Hepatic apoptosis (Figure 2B ) was significantly reduced in the CC10 group.", "Hepatic apoptosis (Figure 2B ) was significantly reduced in the CC10 group. We and collaborators have a long standing interest in studying the role of fgl2 in viral hepatitis. Fgl2 has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports.", "Fgl2 has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports. We have provided liver pathology figures and liver function for MHV-3 infected mice with a fgl2 gene knockout as shown in Supplementary Figure 1 . The data was comparable with previous reports from our center and collaborators.", "The data was comparable with previous reports from our center and collaborators. From this current study we shown that CC10 plays a protective role in liver damage.To study the related molecules of CC10 in MHV-3-induced FH mice, we evaluated whether there was crosstalk between Fgl2 and CC10. We found that the expression of Fgl2 in the liver of mice was reduced 72 h after MHV-3 infection and treatment with CC10 protein (Figures 3A,B) .", "We found that the expression of Fgl2 in the liver of mice was reduced 72 h after MHV-3 infection and treatment with CC10 protein (Figures 3A,B) . Furthermore, fibrin deposition, an indicator of liver injury associated with Fgl2 expression in FH, was also decreased in the livers of CC10-treated mice compared to that in controls (Figure 3C ). This indicates that CC10 treatment reduced liver injury after viral infection by inhibiting Fgl2 expression.", "This indicates that CC10 treatment reduced liver injury after viral infection by inhibiting Fgl2 expression. We examined the effect of increasing doses of CC10 protein (0, 50, 150, and 300 ng/mL) on IFN-γ-induced Fgl2 expression in THP-1 cells. CC10 treatment showed a 10.1% decrease in THP-1 cells compared to that in control after stimulation with 10 ng/mL IFN-γ for 12 h. CC10 protein inhibited Fgl2 expression between doses of 0 ng/mL and 300 ng/mL (Figure 4A ).", "CC10 treatment showed a 10.1% decrease in THP-1 cells compared to that in control after stimulation with 10 ng/mL IFN-γ for 12 h. CC10 protein inhibited Fgl2 expression between doses of 0 ng/mL and 300 ng/mL (Figure 4A ). In particular, 150 ng/mL CC10 protein had the strongest inhibitory effect on Fgl2 expression among the doses, and we chose this dose for the following experiments. We explored the effect of different time points of stimulation with a concentration of 150 ng/mL CC10 protein.", "We explored the effect of different time points of stimulation with a concentration of 150 ng/mL CC10 protein. After stimulation with CC10 protein for 6, 12, and 24 h compared to the PBS control, the strongest inhibitory effect on Fgl2 expression was noted at 12 h; hence, we chose this time point for the following studies ( Figure 4B ). An increasing number of studies suggest that macrophages are the primary source of Fgl2.", "An increasing number of studies suggest that macrophages are the primary source of Fgl2. In order to ascertain that CC10 has a direct effect on macrophages, we treated THP-1 cells with recombinant CC10 and assessed the expression of Fgl2. Unlike in controls, IFN-γ induced a significant increase in Fgl2 expression.", "Unlike in controls, IFN-γ induced a significant increase in Fgl2 expression. This effect was attenuated when cells were treated with CC10 protein (Figures 4C,D) , revealing that CC10 directly reduces the levels of Fgl2 in macrophages. To further explore the possibility that CC10 protein directly acts on macrophages, we infected murine PEMs with MHV-3 in the presence of recombinant CC10 and determined Fgl2 expression.", "To further explore the possibility that CC10 protein directly acts on macrophages, we infected murine PEMs with MHV-3 in the presence of recombinant CC10 and determined Fgl2 expression. Compared to levels in the controls, MHV-3infected macrophages exhibited a significant increase in Fgl2 production, and this effect was abolished by using CC10 protein (Figures 5A,B) , indicating that CC10 directly modulates Fgl2 production in macrophages. In order to determine genes that were downregulated after stimulation by CC10 protein, we used DNA microarray analysis to screen for differentially expressed genes.", "In order to determine genes that were downregulated after stimulation by CC10 protein, we used DNA microarray analysis to screen for differentially expressed genes. THP-1 cells were cultured and PMA was added to induce differentiation into macrophages. The production of Fgl2 was stimulated by IFNγ.", "The production of Fgl2 was stimulated by IFNγ. The experimental group was treated with CC10 protein for microarray detection of differentially expressed genes. The results showed that the most obviously downregulated genes were UBE2W, HECTD1, MIR612, ATRX, SOX4, HBP1, and Fgl2 (Supplementary Table 1) .", "The results showed that the most obviously downregulated genes were UBE2W, HECTD1, MIR612, ATRX, SOX4, HBP1, and Fgl2 (Supplementary Table 1) . And then these genes were tested by qPCR. However, UBE2W, HECTD1, MIR612, ATRX, and SOX4 was not differentially expressed by qPCR, while HBP1 and fgl2 were still down-regulated genes.", "However, UBE2W, HECTD1, MIR612, ATRX, and SOX4 was not differentially expressed by qPCR, while HBP1 and fgl2 were still down-regulated genes. DNA microarray analysis identified HBP1 as a down-regulated gene involved in the pathological processes of the regulation of CC10. Recently, very limited studies have explored the role of HBP1 in FH.", "Recently, very limited studies have explored the role of HBP1 in FH. Nevertheless, the mechanistic functions of HBP1 in FH remain largely unexplored. Therefore, we selected this gene for further study.", "Therefore, we selected this gene for further study. qPCR analysis confirmed that mRNA levels of HBP1 were significantly decreased in THP-1 cells after CC10\n\nprotein stimulation compared to that in the PBS control group (Figure 6A ). We knocked down HBP1 using HBP1-siRNA. Then, transfection of HBP1-SiRNA into HUVECs was detected by qPCR and western-blotting methods.", "Then, transfection of HBP1-SiRNA into HUVECs was detected by qPCR and western-blotting methods. As expected, HBP1 knockdown led to significantly decreased expression of HBP1 (Figures 6B,C) . Furthermore, HBP1 knockdown impaired expression of Fgl2 (Figure 6D ), suggesting that HBP1 was able to activate Fgl2. HBP1-SiRNA was used to transfect HUVECs.", "HBP1-SiRNA was used to transfect HUVECs. Then, IFN-γ was added to induce the expression of Fgl2 followed by stimulation with CC10 protein (150 ng/ml) after 2 h. Finally, we explored the expression of Fgl2 by qPCR. The results showed that HBP1-SiRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in HUVECs (Figure 7) .", "The results showed that HBP1-SiRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in HUVECs (Figure 7) . That is to say, CC10 could suppress Fgl2 expression in macrophages. Such an effect may be mediated by the transcription factor HBP1. It is well-known that CC10 protein can suppress the immune response.", "It is well-known that CC10 protein can suppress the immune response. In animal models of allergic diseases of the respiratory tract, most of evidences confirm this inhibition (26) . Its function in FH has not been investigated yet.", "Its function in FH has not been investigated yet. Here, we used a murine FH model established by MHV-3 infection to explore the effects of CC10 in this disease process. To determine the role of CC10 in the pathogenesis of FH, CC10 protein was injected into a mouse FH model established by MHV-3 infection.", "To determine the role of CC10 in the pathogenesis of FH, CC10 protein was injected into a mouse FH model established by MHV-3 infection. MHV-3-induced liver injury in CC10-treated mice occurred rarely and the areas of lesions were much fewer than those in saline-treated control mice. In summary, these results suggested that CC10 could reduce pathological liver damage in this FH model together with lower mortality rates followed by MHV-3 infection.", "In summary, these results suggested that CC10 could reduce pathological liver damage in this FH model together with lower mortality rates followed by MHV-3 infection. MHV-3 induced fulminant viral hepatitis progresses rapidly and infected mice die within 3-5 days. Previous studies suggested fgl2 played a vital role in this process with a 15-40% increase of survival when fgl2 was deleted (12, 15, 27, 28) .", "Previous studies suggested fgl2 played a vital role in this process with a 15-40% increase of survival when fgl2 was deleted (12, 15, 27, 28) . Multiple inflammatory factors or mediators including TNF-α and IFN-γ, IL-1β and C5aR have been demonstrated to promote FH progression with significant discrepancies between liver damage and survival rate (29) (30) (31) (32) , which is accordant with our observation that CC10 substantially alleviated liver injury though survival rate improved mildly. The survival rate based on hours may be more accurate to examine the effect of CC10 on FH.", "The survival rate based on hours may be more accurate to examine the effect of CC10 on FH. It is speculated that fgl2 can mediate lethality in MHV-3-induced FH. This is due to the fact that fgl2 induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity (15) .", "This is due to the fact that fgl2 induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity (15) . To determine whether the tissue necrosis was mediated by Fgl2 in CC10-treated mice following infection, Fgl2 expression was observed. Results suggested that the expression of Fgl2 was significantly increased in MHV-3-induced FH mice and CC10 treatment significantly reduced the production of Fgl2 in the infected liver and serum.", "Results suggested that the expression of Fgl2 was significantly increased in MHV-3-induced FH mice and CC10 treatment significantly reduced the production of Fgl2 in the infected liver and serum. In addition, decreased fibrinogen deposition was also observed in the livers of CC10-treated mice. Therefore, our research results strongly clarify that the lower mortality of CC10-treated mice after MHV-3 infection is due to the lower levels of Fgl2 and decreased fibrinogen deposition.", "Therefore, our research results strongly clarify that the lower mortality of CC10-treated mice after MHV-3 infection is due to the lower levels of Fgl2 and decreased fibrinogen deposition. Indeed, it has been reported that Fgl2 is expressed on macrophages, and the expression of Fgl2 is believed to be induced by IFN-γ and TNF-α (22) . Cultured THP-1 cells activated by IFN-γ or IL-2 have been demonstrated, with induction of Fgl2 expression and enhanced activation of human prothrombin (23) .", "Cultured THP-1 cells activated by IFN-γ or IL-2 have been demonstrated, with induction of Fgl2 expression and enhanced activation of human prothrombin (23) . Therefore, in this study, we explored this cell line to investigate the modulation of CC10 on Fgl2. Surprisingly, we found that CC10 directly inhibited IFN-γ-induced Fgl2 expression in THP-1 cells.", "Surprisingly, we found that CC10 directly inhibited IFN-γ-induced Fgl2 expression in THP-1 cells. As we know, IFN-γ has proved to be the main cytokine that leads to the development and progression of FH. Also, it was shown that IFN-γ might exert its own proinflammatory biological function through enhancing Fgl2 expression.", "Also, it was shown that IFN-γ might exert its own proinflammatory biological function through enhancing Fgl2 expression. Therefore, in our study, CC10 might counter the effect of IFN-γ in the setting of FH, which substantiates its role in FH. These results demonstrated that CC10 regulates the expression of Fgl2 in macrophages.", "These results demonstrated that CC10 regulates the expression of Fgl2 in macrophages. In the current study, we used co-immunoprecipitation to analyze binding between CC10 and Fgl2. In this study, we investigated possible protein-protein interactions between CC10 and Fgl2 in vitro. The Chinese hamster ovary (CHO) cells transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2.", "The Chinese hamster ovary (CHO) cells transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2. Cellular proteins were immunoprecipitated with anti-CC10 antibody or anti-Fgl2 antibody. Immunoblotting was performed with anti-Fgl2 and anti-CC10 antibodies.", "Immunoblotting was performed with anti-Fgl2 and anti-CC10 antibodies. Immunoprecipitation of protein extracts from pcDNA 3.1-CC10 and pcDNA3.1-Fgl2 co-transfected CHO cells with anti-Fgl2 or anti-CC10 antibody followed by western blotting with Fgl2 and CC10 antibodies indicated that CC10 did not co-immunoprecipitate with Fgl2, showing that there is no direct relationship between CC10 and Fgl2 (data not shown). The results showed that CC10 has no direct interaction with Fgl2.", "The results showed that CC10 has no direct interaction with Fgl2. From our previous study the gene of fgl2 contributed profoundly in MHV-3 induced fulminant hepatitis and is extensively expressed in macrophages and endothelium (12, 33) . Our microarray indicated a CC10 down-regulated fgl2 expression and this is further confirmed by qPCR and Western blotting in vivo (peritoneal macrophages) and in vitro (THP-1, macrophage cell line).", "Our microarray indicated a CC10 down-regulated fgl2 expression and this is further confirmed by qPCR and Western blotting in vivo (peritoneal macrophages) and in vitro (THP-1, macrophage cell line). Therefore, it is reasonable to focus on macrophages to display the effect of CC10 on fgl2 expression and eventually mice survival. We entirely agree there may be other possibilities for a protective effect of CC10 to contribute to the disease process.", "We entirely agree there may be other possibilities for a protective effect of CC10 to contribute to the disease process. This is worth further studies. The potential receptor of CC10 has not been revealed yet. Our previous study have demonstrated that CC10 have effect of dendritic cells in allergic rhinitis (34) .", "Our previous study have demonstrated that CC10 have effect of dendritic cells in allergic rhinitis (34) . In this research, we evaluated the effect of CC10 on macrophages functions and found Fgl2 was substantially down-regulated upon CC10 treatment, therefore, we speculate that potential CC10 receptor may be also expressed on macrophages. The potential target of CC10 on other immune cells cannot be excluded.", "The potential target of CC10 on other immune cells cannot be excluded. DNA microarray analysis is one of the most powerful approaches for the potential identification of unexpected genes involved in pathogenic processes. By using this approach, HMGbox transcription factor 1 (HBP1) was found to be one of the most downregulated genes after CC10 treatment of THP-1 cells.", "By using this approach, HMGbox transcription factor 1 (HBP1) was found to be one of the most downregulated genes after CC10 treatment of THP-1 cells. HBP1 is a well-described transcriptional repressor that modulates expression of genes involved in cell cycle progression. In a recent study, it was found that HBP1 is a direct target of miR-21 and confirmed that HBP1 modulates the inhibitory function of miR-21-ASO in hepatosteatosis and carcinogenesis simultaneously (23) .", "In a recent study, it was found that HBP1 is a direct target of miR-21 and confirmed that HBP1 modulates the inhibitory function of miR-21-ASO in hepatosteatosis and carcinogenesis simultaneously (23) . HBP1 is an endogenous inhibitor of the Wnt signaling pathway in both normal and cancer cells. The tumor suppressor role of HBP1 has been reported in some malignancies, such as oral cancer and glioma (35) .", "The tumor suppressor role of HBP1 has been reported in some malignancies, such as oral cancer and glioma (35) . However, an association between HBP1 and Fgl2 has not been investigated yet. The current study clearly demonstrated that CC10 protects against MHV-3 induced FH via suppression of Fgl2 expression. Such effects might be mediated by HBP1.", "Such effects might be mediated by HBP1. However, the functional status of HBP1 in the CC10 pathway requires further research, and such studies are conducting in our laboratory. In conclusion, we demonstrated that CC10 could limit the immunopathological damage in MHV-3-induced FH mice.", "In conclusion, we demonstrated that CC10 could limit the immunopathological damage in MHV-3-induced FH mice. Our results suggest that enhancing CC10 expression by an immunotherapeutic approach might be an effective treatment for FH. HY performed all the described experiments and wrote the manuscript. YL assisted with some experiments, analyzed experimental results, and edited the manuscript.", "YL assisted with some experiments, analyzed experimental results, and edited the manuscript. HW analyzed experimental results. XW reviewed and edited the manuscript. JH, WY, DX, XL, GS, and QN provided experimental help and design." ]

[ "Background: Fulminant hepatitis (FH) is a serious threat to human life, accompanied by massive and rapid necroinflammation. Kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for FH. Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection.", "Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection. Clara cell 10 kDa (CC10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. However, its mechanisms of action and pathogenic roles in other disease are still unclear.", "However, its mechanisms of action and pathogenic roles in other disease are still unclear. In this study, we aimed to determine the role of CC10 in FH and the regulation of Fgl2 by CC10. Methods: A mouse FH model was established by peritoneal injection of MHV-3.", "Methods: A mouse FH model was established by peritoneal injection of MHV-3. The mice received CC10 protein through tail vein injection before viral infection. Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined.", "Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. The regulatory effect of CC10 on Fgl2 expression was investigated using THP-1 cells and mouse peritoneal macrophages in vitro. Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group.", "Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group. Meanwhile, the levels of ALT and AST in serum were significantly decreased and liver damage was reduced. Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein.", "Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein. In vitro, CC10 was found to significantly inhibit the expression of Fgl2 in IFN-γ-treated THP-1 cells and MHV-3-infected mouse peritoneal macrophages by western blot and real-time PCR. However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation.", "However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation. Microarray investigations suggested that HMG-box transcription factor 1 (HBP1) was significantly low in CC10-treated and IFN-γ-primed THP-1 cells. HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs).", "HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs). Conclusion:CC10 protects against MHV-3-induced FH via suppression of Fgl2 expression in macrophages. Such effects may be mediated by the transcription factor HBP1.", "Such effects may be mediated by the transcription factor HBP1. Text: Fulminant hepatitis (FH) is a serious life-threatening disease characterized by massive hepatocyte necrosis, severe liver damage, and high mortality. The underlying mechanisms and the pathogenesis of FH are not clear.", "The underlying mechanisms and the pathogenesis of FH are not clear. However, accumulating evidence suggests that, regardless of the pathogenesis of FH, the host's inflammatory responses contribute to liver microcirculatory disorders and injuries. Accordingly, It has been shown that immune cell activation and inflammatory cytokines play an important role in FH (1) .", "Accordingly, It has been shown that immune cell activation and inflammatory cytokines play an important role in FH (1) . In recent years, our laboratory has conducted extensive research on the pathogenesis of FH and found that immune cells play a key role in it. Kupffer cells, natural killer (NK) cells (2, 3) , cytotoxic T-lymphocytes (CTLs), and double negative T-cells (DNT) (4) (5) (6) in liver and the cytokines that are produced by these cells cause liver damage.", "Kupffer cells, natural killer (NK) cells (2, 3) , cytotoxic T-lymphocytes (CTLs), and double negative T-cells (DNT) (4) (5) (6) in liver and the cytokines that are produced by these cells cause liver damage. Prothrombinase Fgl2 belongs to the fibrinogen superfamily and is produced by activated macrophages or endothelial cells, transforming prothrombin directly into thrombin, so as to quickly initiate the process of coagulation. This promotes the conversion of fibrinogen into fibrin, resulting in thrombosis (7) (8) (9) (10) (11) (12) .", "This promotes the conversion of fibrinogen into fibrin, resulting in thrombosis (7) (8) (9) (10) (11) (12) . Our study found that Fgl2 was highly expressed in peripheral blood mononuclear cells (PBMCs) and in liver tissue of humans or mice with severe viral hepatitis, and was positively related to the severity of the disease (13, 14) . Gene therapy targeting Fgl2 silencing showed that the survival rate of fulminant hepatitis mice increased from 0 to 33.3% (15) .", "Gene therapy targeting Fgl2 silencing showed that the survival rate of fulminant hepatitis mice increased from 0 to 33.3% (15) . Thus far, the discovery and related research involving Fgl2 have provided new insights into the molecular mechanism of hepatocyte necrosis in FH. In view of the important role of Fgl2 in severe viral hepatitis, investigations concerning the regulation of Fgl2 will be beneficial in the search for new strategies for treatment of severe hepatitis.", "In view of the important role of Fgl2 in severe viral hepatitis, investigations concerning the regulation of Fgl2 will be beneficial in the search for new strategies for treatment of severe hepatitis. Clara cell 10 kDa protein (CC10), also considered to be uteroglobin, Clara cell secretory protein, is one of members of secretoglobin superfamily. Expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world (16) .", "Expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world (16) . CC10 has immunomodulatory and anti-inflammatory effects. Compared to wild-type mice, CC10-knockout mice exhibited excessive airway inflammation Abbreviations: FH, fulminant hepatitis; MHV-3, murine hepatitis virus strain 3; Fgl2, Fibrinogen-like protein 2; CC10, Clara cell 10 KDa protein; ALF, acute liver failure; PFU, plaque-forming units; PBS, phosphate-buffered saline; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCA, pro-coagulant activity; HRP, horseradish peroxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.", "Compared to wild-type mice, CC10-knockout mice exhibited excessive airway inflammation Abbreviations: FH, fulminant hepatitis; MHV-3, murine hepatitis virus strain 3; Fgl2, Fibrinogen-like protein 2; CC10, Clara cell 10 KDa protein; ALF, acute liver failure; PFU, plaque-forming units; PBS, phosphate-buffered saline; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCA, pro-coagulant activity; HRP, horseradish peroxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. caused by allergic reaction and bacterial and viral infections (17) . Reduced levels of CC10 are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis (18) (19) (20) (21) .", "Reduced levels of CC10 are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis (18) (19) (20) (21) . Previous studies and published articles show that CC10 protein can not only inhibit Th17 cell responses by inhibiting expression of related molecules of dendritic cells and cytokines in mice with allergic rhinitis, but also can inhibit chitosan-3 like protein 1 (22, 23) . Moreover, CC10 inhibits the expression of an important immune regulator, osteopontin (OPN), in models of allergic rhinitis (21) .", "Moreover, CC10 inhibits the expression of an important immune regulator, osteopontin (OPN), in models of allergic rhinitis (21) . In this study, we investigated the role of CC10 in hepatitis virus strain 3 (MHV-3)-induced FH in mice and explored whether CC10 protein could regulate Fgl2 in the disease process. Female BALB/cJ mice (Shanghai Shilaike Animal Seed Center, Shanghai, China), 6-8 weeks of age, with a body weight of 18.0-20.0 g, were kept in Tongji Hospital with food and water.", "Female BALB/cJ mice (Shanghai Shilaike Animal Seed Center, Shanghai, China), 6-8 weeks of age, with a body weight of 18.0-20.0 g, were kept in Tongji Hospital with food and water. Mice were divided into two groups: CC10 group (experimental group) and phosphate-buffered saline (PBS) group (control group). This study was carried out in accordance with the recommendations of the guidelines of the National Institutes of Health and the Animal Experiment Committee of Tongji hospital.", "This study was carried out in accordance with the recommendations of the guidelines of the National Institutes of Health and the Animal Experiment Committee of Tongji hospital. This study was reviewed and approved by the Animal Experiment Committee of Tongji hospital. The human monocyte cell line THP-1 was purchased from the Cell Institute of the Chinese Academy of Sciences (Shanghai, China).", "The human monocyte cell line THP-1 was purchased from the Cell Institute of the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from the Biology Treasure Center of Wuhan University, China. The Chinese hamster ovary (CHO) cell line was acquired from the typical culture preservation commission cell bank, the Chinese Academy of Sciences (Shanghai, China).", "The Chinese hamster ovary (CHO) cell line was acquired from the typical culture preservation commission cell bank, the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) and CHO cells were cultured in Dulbecco's modified Eagle's medium (DMEM), and THP-1 cells were maintained in RPMI 1,640 containing 10% heat inactivated fetal bovine serum (FBS, Gibco Life Technologies, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin and cultured at 37 • C, 50 mL/L CO 2 and 95% humidity. Peritoneal exudative macrophages (PEMs) were obtained from BALB/cJ mice.", "Peritoneal exudative macrophages (PEMs) were obtained from BALB/cJ mice. Cells were resuspended in RPMI 1,640 supplemented with 10% FBS at 1-2 × 10 6 cells/mL in a 6-well plate and incubated for 4 h. They were then washed with RPMI 1640 medium and non-adherent cells discarded. The adherent cells were macrophages and were incubated for a further 12 h. Peritoneal exudative macrophages (PEMs) were divided into two groups.", "The adherent cells were macrophages and were incubated for a further 12 h. Peritoneal exudative macrophages (PEMs) were divided into two groups. One group was supplemented with CC10 protein (150 ng/mL) and in the other group, PBS was added. After 2 h of stimulation, 1,000 plaque forming units (PFUs) of MHV-3 was added to the cells, which were then cultured for 4 h. Peritoneal exudative macrophages (PEMs) were harvested and lysed for real-time PCR and western blotting analysis.", "After 2 h of stimulation, 1,000 plaque forming units (PFUs) of MHV-3 was added to the cells, which were then cultured for 4 h. Peritoneal exudative macrophages (PEMs) were harvested and lysed for real-time PCR and western blotting analysis. Cell apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method with a TUNEL apoptosis detection kit (Roche, Switzerland). Briefly, 5 µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase K for 30 min at 37 • C. After stopping the proteinase K digestion reaction with PBS, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dUTP at a ratio of 2:29, respectively), for 2 h at 37 • C in an immunohistochemistry wet box.", "Briefly, 5 µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase K for 30 min at 37 • C. After stopping the proteinase K digestion reaction with PBS, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dUTP at a ratio of 2:29, respectively), for 2 h at 37 • C in an immunohistochemistry wet box. Following washing and blocking, each section was supplemented with reagent (converter-POD) to cover the tissues and incubated for 30 min at 37 • C in a wet box. Then, the liver tissue sections were washed with PBS, and colored with diaminobenzidine (DAB) subsequently.", "Then, the liver tissue sections were washed with PBS, and colored with diaminobenzidine (DAB) subsequently. Hepatocytes with nucleus stained brownish yellow were considered to be apoptotic cells. The expression of Fgl2 on THP-1 cells was measured by flow cytometry (BD FACS Canto II, USA).", "The expression of Fgl2 on THP-1 cells was measured by flow cytometry (BD FACS Canto II, USA). Briefly, cells (2 × 10 5 per tube) were incubated with Human TruStrain FcX (Fc Receptor Blocking solution, BioLegend, USA) for 10 min at room temperature and then incubated in the dark with mouse anti-Fgl2 antibody (1:100, Abnova,) or normal goat serum (an isotype control) at 4 • C for 40 min. Cells were washed with PBS and incubated in the dark with PE-conjugated goat anti-mouse IgG antibody (1:50, BioLegend, USA) at 4 • C for 30 min.", "Cells were washed with PBS and incubated in the dark with PE-conjugated goat anti-mouse IgG antibody (1:50, BioLegend, USA) at 4 • C for 30 min. Cells were then washed with PBS and resuspended in 300 µL PBS for study. Liver slices were fixed in 4% paraformaldehyde and then embedded in paraffin.", "Liver slices were fixed in 4% paraformaldehyde and then embedded in paraffin. Immunohistochemistry of liver tissues was performed using SP-9001 SPlink Detection Kits (Biotin-Streptavidin HRP Detection Systems) (ZSGB-BIO, Beijing, China) according to the manufacturer's instructions. For immunohistochemistry staining, the expression of Fgl2, fibrinogen, Fas and TNF-receptor 1 in mouse liver tissues was detected with polyclonal rabbit anti-mouse Fgl2 antibody (1:100, Proteintech, USA), polyclonal rabbit anti-mouse fibrinogen antibody (1:1,000, Abcam, EngLand), polyclonal rabbit antimouse Fas antibody (1:50, Abcam, EngLand), and polyclonal rabbit anti-mouse TNF-receptor 1 antibody (1:500, Abcam, EngLand), respectively.", "For immunohistochemistry staining, the expression of Fgl2, fibrinogen, Fas and TNF-receptor 1 in mouse liver tissues was detected with polyclonal rabbit anti-mouse Fgl2 antibody (1:100, Proteintech, USA), polyclonal rabbit anti-mouse fibrinogen antibody (1:1,000, Abcam, EngLand), polyclonal rabbit antimouse Fas antibody (1:50, Abcam, EngLand), and polyclonal rabbit anti-mouse TNF-receptor 1 antibody (1:500, Abcam, EngLand), respectively. After incubation with an horseradish peroxidase (HRP)-labeled goat IgG fraction to rabbit IgG Fc, the target protein was detected using a DAB kit (ZSGB-BIO, Beijing, China). The slides were then counterstained with hematoxylin and visualized under a microscope (Olympus, Tokyo, Japan).", "The slides were then counterstained with hematoxylin and visualized under a microscope (Olympus, Tokyo, Japan). Liver tissue and cells were homogenized in RIPA lysis buffer with phenyl methane sulfonyl fluoride (PMSF) protease inhibitor. Protein lysates were separated by SDS-PAGE, and western blotting was performed using a monoclonal mouse antihuman/mouse Fgl2 (1:750, Abnova), a monoclonal mouse antihuman HBP1 (1:100, Santa Cruz, USA), and a monoclonal rabbit anti-human/mouse β-actin (1:1,000, Cell Signaling Technology, USA).", "Protein lysates were separated by SDS-PAGE, and western blotting was performed using a monoclonal mouse antihuman/mouse Fgl2 (1:750, Abnova), a monoclonal mouse antihuman HBP1 (1:100, Santa Cruz, USA), and a monoclonal rabbit anti-human/mouse β-actin (1:1,000, Cell Signaling Technology, USA). Liver tissues were collected from MHV-3-infected BALB/cJ mice at 72 h, and total RNA was extracted using Trizol Reagent (Invitrogen, USA) and then reverse transcribed into cDNA by using ReverTra Ace qPCR RT kit (TOYOBO, Japan). The cDNA was then amplified by RT-PCR by using Dream Taq Green PCR Master Mix (2 ×) (Thermo Scientific, USA).", "The cDNA was then amplified by RT-PCR by using Dream Taq Green PCR Master Mix (2 ×) (Thermo Scientific, USA). Realtime quantitative PCR (qPCR) with SYBR Green Real-time PCR Master Mix (TOYOBO, Japan) was performed using a CFX96 real-time PCR detection system (Bio-Rad, USA) and mRNA levels were normalized with reference to those of the house keeping gene GAPDH. Primer sequences for qPCR amplification were as follows: mTNF-α forward, 5 ′ -TTT GAG ATC CAT GCC GTT GG-3 ′ ; mTNF-α reverse, 5 ′ -GCCA CCA CGC TCT TCT GT-3 ′ ; mIL-1β forward, 5 ′ -TGT AAT GAA AGA CGG CAC ACC-3 ′ ; mIL-1β reverse, 5 ′ -TCT TCT TTG GGT ATT GCT TGG-3 ′ .", "Primer sequences for qPCR amplification were as follows: mTNF-α forward, 5 ′ -TTT GAG ATC CAT GCC GTT GG-3 ′ ; mTNF-α reverse, 5 ′ -GCCA CCA CGC TCT TCT GT-3 ′ ; mIL-1β forward, 5 ′ -TGT AAT GAA AGA CGG CAC ACC-3 ′ ; mIL-1β reverse, 5 ′ -TCT TCT TTG GGT ATT GCT TGG-3 ′ . mFgl2 forward, 5 ′ -GCC AAA TGT GAG TCC CTG GAA-3 ′ ; mFgl2 reverse, 5 ′ -TTC CAC CCA AGA GCA CGT TTA AG-3 ′ ; hFgl2 forward 5 ′ -ACA GTT CAG GCT GGT GGT-3 ′ ; hFgl2 reverse, 5 ′ -GGC TTA AAG TGC TTG GGT-3 ′ ; HBP1 forward, 5 ′ -TGA AGC AGA AGC TGG GAGT-3 ′ ; HBP1 reverse,\n\nTHP-1 cells were treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma, USA) for 48 h to induce differentiation toward adherent macrophage-like cells as reported previously (24) . The CC10 group was supplemented with CC10 protein (150 ng/ml).", "The CC10 group was supplemented with CC10 protein (150 ng/ml). After 2 h of stimulation, IFN-γ (10 ng/ml) was added to these cells, which were then cultured for 12 h before they were collected for western blotting and real-time PCR studies. The Chinese hamster ovary (CHO) cells were cultured in 10 cm cell culture dishes with DMEM supplemented with 10% FBS until 80-90% confluence.", "The Chinese hamster ovary (CHO) cells were cultured in 10 cm cell culture dishes with DMEM supplemented with 10% FBS until 80-90% confluence. Next, 12 µg pcDNA3.1-hFgl2 (constructed in our lab) was mixed with 12 µg pcDNA3.1-hCC10 in serumfree DMEM. The mixture was then combined with Lipofectamine 2,000 (Invitrogen, USA) and mixed gently.", "The mixture was then combined with Lipofectamine 2,000 (Invitrogen, USA) and mixed gently. After incubation at 27 • C for 20 min, the solution was added to CHO cells and incubated at 37 • C in 5% CO 2 . Four to Six hour after transfection, the medium was removed and fresh medium containing 10% FBS was added.", "Four to Six hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, the cells were collected for co-immunoprecipitation analysis to evaluate the interaction of CC10 with Fgl2. Both HUVEC and THP-1 cells express fgl2.", "Both HUVEC and THP-1 cells express fgl2. However, in the transfection experiments, it is difficult to transfect the THP-1 cells with siRNA, so we use HUVEC instead of THP-1. Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in FIGURE 1 | CC10 protein increased survival rate and reduced liver damage in mice.", "Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in FIGURE 1 | CC10 protein increased survival rate and reduced liver damage in mice. (A) The survival rate of CC10 group is higher than the control group comprised of MHV-3-infected BALB/cJ mice treated with saline. CC10 protein (2 µg) or saline were injected into mice by tail vein.", "CC10 protein (2 µg) or saline were injected into mice by tail vein. BALB/cJ mice then received 100 PFU of MHV-3 intraperitoneally 24 h later to develop fulminant viral hepatitis. Then, CC10 protein (2 µg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later.", "Then, CC10 protein (2 µg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later. The survival rate was observed for 10 days (n = 24/group). Representative data from three independent experiments are shown. The survival curve was analyzed by using the Log-Rank Test.", "The survival curve was analyzed by using the Log-Rank Test. ***P < 0.001 compared with saline group. (B) Histopathology of liver tissues (H&E staining; original magnification, ×400, n = 5/group) at 72 h post-MHV-3 infection was evaluated in the two groups of MHV-3-infected BALB/cJ mice.", "(B) Histopathology of liver tissues (H&E staining; original magnification, ×400, n = 5/group) at 72 h post-MHV-3 infection was evaluated in the two groups of MHV-3-infected BALB/cJ mice. Livers were collected from saline-treated (a) and CC10-treated (b) BALB/cJ mice at 72 h after MHV-3 infection. Arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation.", "Arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation. (C) Effect of CC10 on serum ALT and AST levels (n = 6-8/group). Values represent means and standard error of three independent experiments performed in triplicate. **P < 0.01 compared with the saline group.", "**P < 0.01 compared with the saline group. six-well plates with DMEM supplemented with 10% FBS until 70-80% confluence. 50 pmol HBP1-siRNA was mixed with 125 µl serum-free DMEM. Two microliter Lipofectamine 2,000 was gently mixed with serum-free DMEM.", "Two microliter Lipofectamine 2,000 was gently mixed with serum-free DMEM. After incubation at 27 • C for 5 min, the solution was added to HUVECs and incubated at 37 • C. Four hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, cells were collected for real-time PCR and western blot analysis to evaluate the effects of HBP1 on Fgl2.", "At 48 h after transfection, cells were collected for real-time PCR and western blot analysis to evaluate the effects of HBP1 on Fgl2. At 24 h after transfection, the CC10 group was supplemented with the CC10 protein (150 ng/mL). After 4 h of stimulation, IFN-γ (10 ng/mL) was added to these cells.", "After 4 h of stimulation, IFN-γ (10 ng/mL) was added to these cells. These cells were then cultured for 24 h before they were harvested for real-time PCR studies to evaluate the effects of CC10 on Fgl2 by HBP1. Negative control was used as a control.", "Negative control was used as a control. To detect whether there was a potential interaction between CC10 protein and Fgl2, CHO cells were transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2 for 48 h. Cells transfected with empty plasmid pcDNA3.1 (mock) were used as negative controls for CC10 gene transfection. Immunoprecipitation and immunoblotting were performed by using Pierce Co-Immunoprecipitation Kit (Pierce, USA).", "Immunoprecipitation and immunoblotting were performed by using Pierce Co-Immunoprecipitation Kit (Pierce, USA). Total cell proteins were extracted as previously described (25) . The proteins were immunoprecipitated by mouse anti-human Fgl2 antibody (1:500, Abnova).", "The proteins were immunoprecipitated by mouse anti-human Fgl2 antibody (1:500, Abnova). For co-immunoprecipitation experiments, western blotting was performed using both rat anti-human uteroglobin/SCGB1A1 Antibody (1:750, R&D, USA) Frontiers in Immunology | and mouse anti-human Fgl2 antibody (1:500, Abnova). Control isotype rat IgG1 was used as a negative control for primary antibodies.", "Control isotype rat IgG1 was used as a negative control for primary antibodies. The human CC10 coding region gene, including a 389 bp sequence, was amplified from homogenized human turbinate tissue by RT-PCR. In this study, the sequences of PCR primers for CC10 were as follows: hCC10-forward, 5 ′ -CCC TCC ACC ATG AAA CTCG-3 ′ ; hCC10-reverse, 5 ′ -TGA GAT GCT TGT GGT TTA TTG AAG-3 ′ .", "In this study, the sequences of PCR primers for CC10 were as follows: hCC10-forward, 5 ′ -CCC TCC ACC ATG AAA CTCG-3 ′ ; hCC10-reverse, 5 ′ -TGA GAT GCT TGT GGT TTA TTG AAG-3 ′ . The PCR products were cloned into pEASY-T1 cloning vector (TransGEN, Beijing, China) and then subcloned into HindIII/XbaI site of pcDNA3.1 vector (Invitrogen, USA) to form eukaryotic expression plasmids pcDNA3.1-hCC10. Microarray analysis was used to screen changes in genome-wide gene expression patterns in THP-1 cells with or without CC10 protein.", "Microarray analysis was used to screen changes in genome-wide gene expression patterns in THP-1 cells with or without CC10 protein. The changes in over 47,000 human gene expression patterns were assessed using Affymetrix gene microarrays (Human Genome U133 Plus 2.0) (CapitalBio Co.,Ltd., Beijing, China). Three replicates were used for microarrays analysis.", "Three replicates were used for microarrays analysis. Data obtained from the experiments are expressed as means ± SEM. Survival curve comparisons were performed with the Log Rank test. Multiple group analyses for data were evaluated by one-way analyses of variance. Analyses of two group results were performed using Student's t-test to evaluate the statistical significance of differences.", "Analyses of two group results were performed using Student's t-test to evaluate the statistical significance of differences. Values of P < 0.05 indicated significance. To establish an animal model of mouse FH, MHV-3 was injected intraperitoneally to BALB/cJ mice (24 mice/group).", "To establish an animal model of mouse FH, MHV-3 was injected intraperitoneally to BALB/cJ mice (24 mice/group). To further study the role of CC10 in FH, recombinant mouse CC10 protein (2 µg/mouse) or saline was administrated into the tail vein 24 h prior to MHV-3 infection. The same dose of CC10 protein or saline was then administered 24 h later.", "The same dose of CC10 protein or saline was then administered 24 h later. The survival rate of the CC10 and saline groups was observed for 10 days. The results showed that mice in the two groups began to die at 48 h after injection of MHV-3 and exhibited symptoms of horripilation, slow activity, and reduced food consumption.", "The results showed that mice in the two groups began to die at 48 h after injection of MHV-3 and exhibited symptoms of horripilation, slow activity, and reduced food consumption. In the CC10 group 24 mice were alive on day 3 after infection, 4 mice alive on day 4, and 3 of 24 (12.5%) mice recovered from fulminant viral hepatitis. At the same time, in saline treated group, there were 5 mice alive on day 3, 1 mice alive on day 4 after infection, and no mice survived to day 5.", "At the same time, in saline treated group, there were 5 mice alive on day 3, 1 mice alive on day 4 after infection, and no mice survived to day 5. That is to say, the mice in the saline group died within 3 or 4 days. Three of 24 (12.5%) mice of the CC10 group recovered from fulminant viral hepatitis ( Figure 1A) .", "Three of 24 (12.5%) mice of the CC10 group recovered from fulminant viral hepatitis ( Figure 1A) . To better understand the mechanisms underlying the biological effects of the CC10 protein, liver function (ALT and AST levels in serum) and liver histology in mice of MHV-3-infected was performed. Liver tissues were harvested 72 h following MHV-3 infection, and liver histology was detected by H&E staining.", "Liver tissues were harvested 72 h following MHV-3 infection, and liver histology was detected by H&E staining. These results showed that there was substantial inflammatory cell infiltration and widespread necrosis of hepatocytes in the liver tissue of the saline group mice (Figure 1Ba ). There were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the CC10 group 72 h after MHV-3 infection (Figure 1Bb) .", "There were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the CC10 group 72 h after MHV-3 infection (Figure 1Bb) . Serum ALT and AST levels in mice were observed 72 h after MHV-3 infection. The results showed that serum ALT and AST levels in the saline group reached a peak 72 h after MHV-3 infection, but there was no significant increase in the CC10 group compared to the levels in the control group (P < 0.01, Figure 1C) .", "The results showed that serum ALT and AST levels in the saline group reached a peak 72 h after MHV-3 infection, but there was no significant increase in the CC10 group compared to the levels in the control group (P < 0.01, Figure 1C) . These results suggested that CC10 protein has a role in protection against MHV-3-induced liver injury in mice. To further elucidate the mechanisms of reduced liver injury following CC10 protein injection, we investigated the cytokines TNF-α and IL-1β expression.", "To further elucidate the mechanisms of reduced liver injury following CC10 protein injection, we investigated the cytokines TNF-α and IL-1β expression. Because these two cytokines play a crucial role in the liver damage of FH. They are characterized by an increase in apoptosis.", "They are characterized by an increase in apoptosis. Levels of TNF-α and IL-1β in liver tissues were markedly reduced in the CC10 group (as shown in Figure 2A) . Hepatic apoptosis (Figure 2B ) was significantly reduced in the CC10 group.", "Hepatic apoptosis (Figure 2B ) was significantly reduced in the CC10 group. We and collaborators have a long standing interest in studying the role of fgl2 in viral hepatitis. Fgl2 has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports.", "Fgl2 has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports. We have provided liver pathology figures and liver function for MHV-3 infected mice with a fgl2 gene knockout as shown in Supplementary Figure 1 . The data was comparable with previous reports from our center and collaborators.", "The data was comparable with previous reports from our center and collaborators. From this current study we shown that CC10 plays a protective role in liver damage.To study the related molecules of CC10 in MHV-3-induced FH mice, we evaluated whether there was crosstalk between Fgl2 and CC10. We found that the expression of Fgl2 in the liver of mice was reduced 72 h after MHV-3 infection and treatment with CC10 protein (Figures 3A,B) .", "We found that the expression of Fgl2 in the liver of mice was reduced 72 h after MHV-3 infection and treatment with CC10 protein (Figures 3A,B) . Furthermore, fibrin deposition, an indicator of liver injury associated with Fgl2 expression in FH, was also decreased in the livers of CC10-treated mice compared to that in controls (Figure 3C ). This indicates that CC10 treatment reduced liver injury after viral infection by inhibiting Fgl2 expression.", "This indicates that CC10 treatment reduced liver injury after viral infection by inhibiting Fgl2 expression. We examined the effect of increasing doses of CC10 protein (0, 50, 150, and 300 ng/mL) on IFN-γ-induced Fgl2 expression in THP-1 cells. CC10 treatment showed a 10.1% decrease in THP-1 cells compared to that in control after stimulation with 10 ng/mL IFN-γ for 12 h. CC10 protein inhibited Fgl2 expression between doses of 0 ng/mL and 300 ng/mL (Figure 4A ).", "CC10 treatment showed a 10.1% decrease in THP-1 cells compared to that in control after stimulation with 10 ng/mL IFN-γ for 12 h. CC10 protein inhibited Fgl2 expression between doses of 0 ng/mL and 300 ng/mL (Figure 4A ). In particular, 150 ng/mL CC10 protein had the strongest inhibitory effect on Fgl2 expression among the doses, and we chose this dose for the following experiments. We explored the effect of different time points of stimulation with a concentration of 150 ng/mL CC10 protein.", "We explored the effect of different time points of stimulation with a concentration of 150 ng/mL CC10 protein. After stimulation with CC10 protein for 6, 12, and 24 h compared to the PBS control, the strongest inhibitory effect on Fgl2 expression was noted at 12 h; hence, we chose this time point for the following studies ( Figure 4B ). An increasing number of studies suggest that macrophages are the primary source of Fgl2.", "An increasing number of studies suggest that macrophages are the primary source of Fgl2. In order to ascertain that CC10 has a direct effect on macrophages, we treated THP-1 cells with recombinant CC10 and assessed the expression of Fgl2. Unlike in controls, IFN-γ induced a significant increase in Fgl2 expression.", "Unlike in controls, IFN-γ induced a significant increase in Fgl2 expression. This effect was attenuated when cells were treated with CC10 protein (Figures 4C,D) , revealing that CC10 directly reduces the levels of Fgl2 in macrophages. To further explore the possibility that CC10 protein directly acts on macrophages, we infected murine PEMs with MHV-3 in the presence of recombinant CC10 and determined Fgl2 expression.", "To further explore the possibility that CC10 protein directly acts on macrophages, we infected murine PEMs with MHV-3 in the presence of recombinant CC10 and determined Fgl2 expression. Compared to levels in the controls, MHV-3infected macrophages exhibited a significant increase in Fgl2 production, and this effect was abolished by using CC10 protein (Figures 5A,B) , indicating that CC10 directly modulates Fgl2 production in macrophages. In order to determine genes that were downregulated after stimulation by CC10 protein, we used DNA microarray analysis to screen for differentially expressed genes.", "In order to determine genes that were downregulated after stimulation by CC10 protein, we used DNA microarray analysis to screen for differentially expressed genes. THP-1 cells were cultured and PMA was added to induce differentiation into macrophages. The production of Fgl2 was stimulated by IFNγ.", "The production of Fgl2 was stimulated by IFNγ. The experimental group was treated with CC10 protein for microarray detection of differentially expressed genes. The results showed that the most obviously downregulated genes were UBE2W, HECTD1, MIR612, ATRX, SOX4, HBP1, and Fgl2 (Supplementary Table 1) .", "The results showed that the most obviously downregulated genes were UBE2W, HECTD1, MIR612, ATRX, SOX4, HBP1, and Fgl2 (Supplementary Table 1) . And then these genes were tested by qPCR. However, UBE2W, HECTD1, MIR612, ATRX, and SOX4 was not differentially expressed by qPCR, while HBP1 and fgl2 were still down-regulated genes.", "However, UBE2W, HECTD1, MIR612, ATRX, and SOX4 was not differentially expressed by qPCR, while HBP1 and fgl2 were still down-regulated genes. DNA microarray analysis identified HBP1 as a down-regulated gene involved in the pathological processes of the regulation of CC10. Recently, very limited studies have explored the role of HBP1 in FH.", "Recently, very limited studies have explored the role of HBP1 in FH. Nevertheless, the mechanistic functions of HBP1 in FH remain largely unexplored. Therefore, we selected this gene for further study.", "Therefore, we selected this gene for further study. qPCR analysis confirmed that mRNA levels of HBP1 were significantly decreased in THP-1 cells after CC10\n\nprotein stimulation compared to that in the PBS control group (Figure 6A ). We knocked down HBP1 using HBP1-siRNA. Then, transfection of HBP1-SiRNA into HUVECs was detected by qPCR and western-blotting methods.", "Then, transfection of HBP1-SiRNA into HUVECs was detected by qPCR and western-blotting methods. As expected, HBP1 knockdown led to significantly decreased expression of HBP1 (Figures 6B,C) . Furthermore, HBP1 knockdown impaired expression of Fgl2 (Figure 6D ), suggesting that HBP1 was able to activate Fgl2. HBP1-SiRNA was used to transfect HUVECs.", "HBP1-SiRNA was used to transfect HUVECs. Then, IFN-γ was added to induce the expression of Fgl2 followed by stimulation with CC10 protein (150 ng/ml) after 2 h. Finally, we explored the expression of Fgl2 by qPCR. The results showed that HBP1-SiRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in HUVECs (Figure 7) .", "The results showed that HBP1-SiRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in HUVECs (Figure 7) . That is to say, CC10 could suppress Fgl2 expression in macrophages. Such an effect may be mediated by the transcription factor HBP1. It is well-known that CC10 protein can suppress the immune response.", "It is well-known that CC10 protein can suppress the immune response. In animal models of allergic diseases of the respiratory tract, most of evidences confirm this inhibition (26) . Its function in FH has not been investigated yet.", "Its function in FH has not been investigated yet. Here, we used a murine FH model established by MHV-3 infection to explore the effects of CC10 in this disease process. To determine the role of CC10 in the pathogenesis of FH, CC10 protein was injected into a mouse FH model established by MHV-3 infection.", "To determine the role of CC10 in the pathogenesis of FH, CC10 protein was injected into a mouse FH model established by MHV-3 infection. MHV-3-induced liver injury in CC10-treated mice occurred rarely and the areas of lesions were much fewer than those in saline-treated control mice. In summary, these results suggested that CC10 could reduce pathological liver damage in this FH model together with lower mortality rates followed by MHV-3 infection.", "In summary, these results suggested that CC10 could reduce pathological liver damage in this FH model together with lower mortality rates followed by MHV-3 infection. MHV-3 induced fulminant viral hepatitis progresses rapidly and infected mice die within 3-5 days. Previous studies suggested fgl2 played a vital role in this process with a 15-40% increase of survival when fgl2 was deleted (12, 15, 27, 28) .", "Previous studies suggested fgl2 played a vital role in this process with a 15-40% increase of survival when fgl2 was deleted (12, 15, 27, 28) . Multiple inflammatory factors or mediators including TNF-α and IFN-γ, IL-1β and C5aR have been demonstrated to promote FH progression with significant discrepancies between liver damage and survival rate (29) (30) (31) (32) , which is accordant with our observation that CC10 substantially alleviated liver injury though survival rate improved mildly. The survival rate based on hours may be more accurate to examine the effect of CC10 on FH.", "The survival rate based on hours may be more accurate to examine the effect of CC10 on FH. It is speculated that fgl2 can mediate lethality in MHV-3-induced FH. This is due to the fact that fgl2 induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity (15) .", "This is due to the fact that fgl2 induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity (15) . To determine whether the tissue necrosis was mediated by Fgl2 in CC10-treated mice following infection, Fgl2 expression was observed. Results suggested that the expression of Fgl2 was significantly increased in MHV-3-induced FH mice and CC10 treatment significantly reduced the production of Fgl2 in the infected liver and serum.", "Results suggested that the expression of Fgl2 was significantly increased in MHV-3-induced FH mice and CC10 treatment significantly reduced the production of Fgl2 in the infected liver and serum. In addition, decreased fibrinogen deposition was also observed in the livers of CC10-treated mice. Therefore, our research results strongly clarify that the lower mortality of CC10-treated mice after MHV-3 infection is due to the lower levels of Fgl2 and decreased fibrinogen deposition.", "Therefore, our research results strongly clarify that the lower mortality of CC10-treated mice after MHV-3 infection is due to the lower levels of Fgl2 and decreased fibrinogen deposition. Indeed, it has been reported that Fgl2 is expressed on macrophages, and the expression of Fgl2 is believed to be induced by IFN-γ and TNF-α (22) . Cultured THP-1 cells activated by IFN-γ or IL-2 have been demonstrated, with induction of Fgl2 expression and enhanced activation of human prothrombin (23) .", "Cultured THP-1 cells activated by IFN-γ or IL-2 have been demonstrated, with induction of Fgl2 expression and enhanced activation of human prothrombin (23) . Therefore, in this study, we explored this cell line to investigate the modulation of CC10 on Fgl2. Surprisingly, we found that CC10 directly inhibited IFN-γ-induced Fgl2 expression in THP-1 cells.", "Surprisingly, we found that CC10 directly inhibited IFN-γ-induced Fgl2 expression in THP-1 cells. As we know, IFN-γ has proved to be the main cytokine that leads to the development and progression of FH. Also, it was shown that IFN-γ might exert its own proinflammatory biological function through enhancing Fgl2 expression.", "Also, it was shown that IFN-γ might exert its own proinflammatory biological function through enhancing Fgl2 expression. Therefore, in our study, CC10 might counter the effect of IFN-γ in the setting of FH, which substantiates its role in FH. These results demonstrated that CC10 regulates the expression of Fgl2 in macrophages.", "These results demonstrated that CC10 regulates the expression of Fgl2 in macrophages. In the current study, we used co-immunoprecipitation to analyze binding between CC10 and Fgl2. In this study, we investigated possible protein-protein interactions between CC10 and Fgl2 in vitro. The Chinese hamster ovary (CHO) cells transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2.", "The Chinese hamster ovary (CHO) cells transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2. Cellular proteins were immunoprecipitated with anti-CC10 antibody or anti-Fgl2 antibody. Immunoblotting was performed with anti-Fgl2 and anti-CC10 antibodies.", "Immunoblotting was performed with anti-Fgl2 and anti-CC10 antibodies. Immunoprecipitation of protein extracts from pcDNA 3.1-CC10 and pcDNA3.1-Fgl2 co-transfected CHO cells with anti-Fgl2 or anti-CC10 antibody followed by western blotting with Fgl2 and CC10 antibodies indicated that CC10 did not co-immunoprecipitate with Fgl2, showing that there is no direct relationship between CC10 and Fgl2 (data not shown). The results showed that CC10 has no direct interaction with Fgl2.", "The results showed that CC10 has no direct interaction with Fgl2. From our previous study the gene of fgl2 contributed profoundly in MHV-3 induced fulminant hepatitis and is extensively expressed in macrophages and endothelium (12, 33) . Our microarray indicated a CC10 down-regulated fgl2 expression and this is further confirmed by qPCR and Western blotting in vivo (peritoneal macrophages) and in vitro (THP-1, macrophage cell line).", "Our microarray indicated a CC10 down-regulated fgl2 expression and this is further confirmed by qPCR and Western blotting in vivo (peritoneal macrophages) and in vitro (THP-1, macrophage cell line). Therefore, it is reasonable to focus on macrophages to display the effect of CC10 on fgl2 expression and eventually mice survival. We entirely agree there may be other possibilities for a protective effect of CC10 to contribute to the disease process.", "We entirely agree there may be other possibilities for a protective effect of CC10 to contribute to the disease process. This is worth further studies. The potential receptor of CC10 has not been revealed yet. Our previous study have demonstrated that CC10 have effect of dendritic cells in allergic rhinitis (34) .", "Our previous study have demonstrated that CC10 have effect of dendritic cells in allergic rhinitis (34) . In this research, we evaluated the effect of CC10 on macrophages functions and found Fgl2 was substantially down-regulated upon CC10 treatment, therefore, we speculate that potential CC10 receptor may be also expressed on macrophages. The potential target of CC10 on other immune cells cannot be excluded.", "The potential target of CC10 on other immune cells cannot be excluded. DNA microarray analysis is one of the most powerful approaches for the potential identification of unexpected genes involved in pathogenic processes. By using this approach, HMGbox transcription factor 1 (HBP1) was found to be one of the most downregulated genes after CC10 treatment of THP-1 cells.", "By using this approach, HMGbox transcription factor 1 (HBP1) was found to be one of the most downregulated genes after CC10 treatment of THP-1 cells. HBP1 is a well-described transcriptional repressor that modulates expression of genes involved in cell cycle progression. In a recent study, it was found that HBP1 is a direct target of miR-21 and confirmed that HBP1 modulates the inhibitory function of miR-21-ASO in hepatosteatosis and carcinogenesis simultaneously (23) .", "In a recent study, it was found that HBP1 is a direct target of miR-21 and confirmed that HBP1 modulates the inhibitory function of miR-21-ASO in hepatosteatosis and carcinogenesis simultaneously (23) . HBP1 is an endogenous inhibitor of the Wnt signaling pathway in both normal and cancer cells. The tumor suppressor role of HBP1 has been reported in some malignancies, such as oral cancer and glioma (35) .", "The tumor suppressor role of HBP1 has been reported in some malignancies, such as oral cancer and glioma (35) . However, an association between HBP1 and Fgl2 has not been investigated yet. The current study clearly demonstrated that CC10 protects against MHV-3 induced FH via suppression of Fgl2 expression. Such effects might be mediated by HBP1.", "Such effects might be mediated by HBP1. However, the functional status of HBP1 in the CC10 pathway requires further research, and such studies are conducting in our laboratory. In conclusion, we demonstrated that CC10 could limit the immunopathological damage in MHV-3-induced FH mice.", "In conclusion, we demonstrated that CC10 could limit the immunopathological damage in MHV-3-induced FH mice. Our results suggest that enhancing CC10 expression by an immunotherapeutic approach might be an effective treatment for FH. HY performed all the described experiments and wrote the manuscript. YL assisted with some experiments, analyzed experimental results, and edited the manuscript.", "YL assisted with some experiments, analyzed experimental results, and edited the manuscript. HW analyzed experimental results. XW reviewed and edited the manuscript. JH, WY, DX, XL, GS, and QN provided experimental help and design." ]

[ "Background: Fulminant hepatitis (FH) is a serious threat to human life, accompanied by massive and rapid necroinflammation. Kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for FH. Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection.", "Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection. Clara cell 10 kDa (CC10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. However, its mechanisms of action and pathogenic roles in other disease are still unclear.", "However, its mechanisms of action and pathogenic roles in other disease are still unclear. In this study, we aimed to determine the role of CC10 in FH and the regulation of Fgl2 by CC10. Methods: A mouse FH model was established by peritoneal injection of MHV-3.", "Methods: A mouse FH model was established by peritoneal injection of MHV-3. The mice received CC10 protein through tail vein injection before viral infection. Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined.", "Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. The regulatory effect of CC10 on Fgl2 expression was investigated using THP-1 cells and mouse peritoneal macrophages in vitro. Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group.", "Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group. Meanwhile, the levels of ALT and AST in serum were significantly decreased and liver damage was reduced. Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein.", "Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein. In vitro, CC10 was found to significantly inhibit the expression of Fgl2 in IFN-γ-treated THP-1 cells and MHV-3-infected mouse peritoneal macrophages by western blot and real-time PCR. However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation.", "However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation. Microarray investigations suggested that HMG-box transcription factor 1 (HBP1) was significantly low in CC10-treated and IFN-γ-primed THP-1 cells. HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs).", "HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs). Conclusion:CC10 protects against MHV-3-induced FH via suppression of Fgl2 expression in macrophages. Such effects may be mediated by the transcription factor HBP1.", "Such effects may be mediated by the transcription factor HBP1. Text: Fulminant hepatitis (FH) is a serious life-threatening disease characterized by massive hepatocyte necrosis, severe liver damage, and high mortality. The underlying mechanisms and the pathogenesis of FH are not clear.", "The underlying mechanisms and the pathogenesis of FH are not clear. However, accumulating evidence suggests that, regardless of the pathogenesis of FH, the host's inflammatory responses contribute to liver microcirculatory disorders and injuries. Accordingly, It has been shown that immune cell activation and inflammatory cytokines play an important role in FH (1) .", "Accordingly, It has been shown that immune cell activation and inflammatory cytokines play an important role in FH (1) . In recent years, our laboratory has conducted extensive research on the pathogenesis of FH and found that immune cells play a key role in it. Kupffer cells, natural killer (NK) cells (2, 3) , cytotoxic T-lymphocytes (CTLs), and double negative T-cells (DNT) (4) (5) (6) in liver and the cytokines that are produced by these cells cause liver damage.", "Kupffer cells, natural killer (NK) cells (2, 3) , cytotoxic T-lymphocytes (CTLs), and double negative T-cells (DNT) (4) (5) (6) in liver and the cytokines that are produced by these cells cause liver damage. Prothrombinase Fgl2 belongs to the fibrinogen superfamily and is produced by activated macrophages or endothelial cells, transforming prothrombin directly into thrombin, so as to quickly initiate the process of coagulation. This promotes the conversion of fibrinogen into fibrin, resulting in thrombosis (7) (8) (9) (10) (11) (12) .", "This promotes the conversion of fibrinogen into fibrin, resulting in thrombosis (7) (8) (9) (10) (11) (12) . Our study found that Fgl2 was highly expressed in peripheral blood mononuclear cells (PBMCs) and in liver tissue of humans or mice with severe viral hepatitis, and was positively related to the severity of the disease (13, 14) . Gene therapy targeting Fgl2 silencing showed that the survival rate of fulminant hepatitis mice increased from 0 to 33.3% (15) .", "Gene therapy targeting Fgl2 silencing showed that the survival rate of fulminant hepatitis mice increased from 0 to 33.3% (15) . Thus far, the discovery and related research involving Fgl2 have provided new insights into the molecular mechanism of hepatocyte necrosis in FH. In view of the important role of Fgl2 in severe viral hepatitis, investigations concerning the regulation of Fgl2 will be beneficial in the search for new strategies for treatment of severe hepatitis.", "In view of the important role of Fgl2 in severe viral hepatitis, investigations concerning the regulation of Fgl2 will be beneficial in the search for new strategies for treatment of severe hepatitis. Clara cell 10 kDa protein (CC10), also considered to be uteroglobin, Clara cell secretory protein, is one of members of secretoglobin superfamily. Expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world (16) .", "Expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world (16) . CC10 has immunomodulatory and anti-inflammatory effects. Compared to wild-type mice, CC10-knockout mice exhibited excessive airway inflammation Abbreviations: FH, fulminant hepatitis; MHV-3, murine hepatitis virus strain 3; Fgl2, Fibrinogen-like protein 2; CC10, Clara cell 10 KDa protein; ALF, acute liver failure; PFU, plaque-forming units; PBS, phosphate-buffered saline; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCA, pro-coagulant activity; HRP, horseradish peroxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.", "Compared to wild-type mice, CC10-knockout mice exhibited excessive airway inflammation Abbreviations: FH, fulminant hepatitis; MHV-3, murine hepatitis virus strain 3; Fgl2, Fibrinogen-like protein 2; CC10, Clara cell 10 KDa protein; ALF, acute liver failure; PFU, plaque-forming units; PBS, phosphate-buffered saline; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCA, pro-coagulant activity; HRP, horseradish peroxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. caused by allergic reaction and bacterial and viral infections (17) . Reduced levels of CC10 are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis (18) (19) (20) (21) .", "Reduced levels of CC10 are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis (18) (19) (20) (21) . Previous studies and published articles show that CC10 protein can not only inhibit Th17 cell responses by inhibiting expression of related molecules of dendritic cells and cytokines in mice with allergic rhinitis, but also can inhibit chitosan-3 like protein 1 (22, 23) . Moreover, CC10 inhibits the expression of an important immune regulator, osteopontin (OPN), in models of allergic rhinitis (21) .", "Moreover, CC10 inhibits the expression of an important immune regulator, osteopontin (OPN), in models of allergic rhinitis (21) . In this study, we investigated the role of CC10 in hepatitis virus strain 3 (MHV-3)-induced FH in mice and explored whether CC10 protein could regulate Fgl2 in the disease process. Female BALB/cJ mice (Shanghai Shilaike Animal Seed Center, Shanghai, China), 6-8 weeks of age, with a body weight of 18.0-20.0 g, were kept in Tongji Hospital with food and water.", "Female BALB/cJ mice (Shanghai Shilaike Animal Seed Center, Shanghai, China), 6-8 weeks of age, with a body weight of 18.0-20.0 g, were kept in Tongji Hospital with food and water. Mice were divided into two groups: CC10 group (experimental group) and phosphate-buffered saline (PBS) group (control group). This study was carried out in accordance with the recommendations of the guidelines of the National Institutes of Health and the Animal Experiment Committee of Tongji hospital.", "This study was carried out in accordance with the recommendations of the guidelines of the National Institutes of Health and the Animal Experiment Committee of Tongji hospital. This study was reviewed and approved by the Animal Experiment Committee of Tongji hospital. The human monocyte cell line THP-1 was purchased from the Cell Institute of the Chinese Academy of Sciences (Shanghai, China).", "The human monocyte cell line THP-1 was purchased from the Cell Institute of the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from the Biology Treasure Center of Wuhan University, China. The Chinese hamster ovary (CHO) cell line was acquired from the typical culture preservation commission cell bank, the Chinese Academy of Sciences (Shanghai, China).", "The Chinese hamster ovary (CHO) cell line was acquired from the typical culture preservation commission cell bank, the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) and CHO cells were cultured in Dulbecco's modified Eagle's medium (DMEM), and THP-1 cells were maintained in RPMI 1,640 containing 10% heat inactivated fetal bovine serum (FBS, Gibco Life Technologies, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin and cultured at 37 • C, 50 mL/L CO 2 and 95% humidity. Peritoneal exudative macrophages (PEMs) were obtained from BALB/cJ mice.", "Peritoneal exudative macrophages (PEMs) were obtained from BALB/cJ mice. Cells were resuspended in RPMI 1,640 supplemented with 10% FBS at 1-2 × 10 6 cells/mL in a 6-well plate and incubated for 4 h. They were then washed with RPMI 1640 medium and non-adherent cells discarded. The adherent cells were macrophages and were incubated for a further 12 h. Peritoneal exudative macrophages (PEMs) were divided into two groups.", "The adherent cells were macrophages and were incubated for a further 12 h. Peritoneal exudative macrophages (PEMs) were divided into two groups. One group was supplemented with CC10 protein (150 ng/mL) and in the other group, PBS was added. After 2 h of stimulation, 1,000 plaque forming units (PFUs) of MHV-3 was added to the cells, which were then cultured for 4 h. Peritoneal exudative macrophages (PEMs) were harvested and lysed for real-time PCR and western blotting analysis.", "After 2 h of stimulation, 1,000 plaque forming units (PFUs) of MHV-3 was added to the cells, which were then cultured for 4 h. Peritoneal exudative macrophages (PEMs) were harvested and lysed for real-time PCR and western blotting analysis. Cell apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method with a TUNEL apoptosis detection kit (Roche, Switzerland). Briefly, 5 µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase K for 30 min at 37 • C. After stopping the proteinase K digestion reaction with PBS, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dUTP at a ratio of 2:29, respectively), for 2 h at 37 • C in an immunohistochemistry wet box.", "Briefly, 5 µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase K for 30 min at 37 • C. After stopping the proteinase K digestion reaction with PBS, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dUTP at a ratio of 2:29, respectively), for 2 h at 37 • C in an immunohistochemistry wet box. Following washing and blocking, each section was supplemented with reagent (converter-POD) to cover the tissues and incubated for 30 min at 37 • C in a wet box. Then, the liver tissue sections were washed with PBS, and colored with diaminobenzidine (DAB) subsequently.", "Then, the liver tissue sections were washed with PBS, and colored with diaminobenzidine (DAB) subsequently. Hepatocytes with nucleus stained brownish yellow were considered to be apoptotic cells. The expression of Fgl2 on THP-1 cells was measured by flow cytometry (BD FACS Canto II, USA).", "The expression of Fgl2 on THP-1 cells was measured by flow cytometry (BD FACS Canto II, USA). Briefly, cells (2 × 10 5 per tube) were incubated with Human TruStrain FcX (Fc Receptor Blocking solution, BioLegend, USA) for 10 min at room temperature and then incubated in the dark with mouse anti-Fgl2 antibody (1:100, Abnova,) or normal goat serum (an isotype control) at 4 • C for 40 min. Cells were washed with PBS and incubated in the dark with PE-conjugated goat anti-mouse IgG antibody (1:50, BioLegend, USA) at 4 • C for 30 min.", "Cells were washed with PBS and incubated in the dark with PE-conjugated goat anti-mouse IgG antibody (1:50, BioLegend, USA) at 4 • C for 30 min. Cells were then washed with PBS and resuspended in 300 µL PBS for study. Liver slices were fixed in 4% paraformaldehyde and then embedded in paraffin.", "Liver slices were fixed in 4% paraformaldehyde and then embedded in paraffin. Immunohistochemistry of liver tissues was performed using SP-9001 SPlink Detection Kits (Biotin-Streptavidin HRP Detection Systems) (ZSGB-BIO, Beijing, China) according to the manufacturer's instructions. For immunohistochemistry staining, the expression of Fgl2, fibrinogen, Fas and TNF-receptor 1 in mouse liver tissues was detected with polyclonal rabbit anti-mouse Fgl2 antibody (1:100, Proteintech, USA), polyclonal rabbit anti-mouse fibrinogen antibody (1:1,000, Abcam, EngLand), polyclonal rabbit antimouse Fas antibody (1:50, Abcam, EngLand), and polyclonal rabbit anti-mouse TNF-receptor 1 antibody (1:500, Abcam, EngLand), respectively.", "For immunohistochemistry staining, the expression of Fgl2, fibrinogen, Fas and TNF-receptor 1 in mouse liver tissues was detected with polyclonal rabbit anti-mouse Fgl2 antibody (1:100, Proteintech, USA), polyclonal rabbit anti-mouse fibrinogen antibody (1:1,000, Abcam, EngLand), polyclonal rabbit antimouse Fas antibody (1:50, Abcam, EngLand), and polyclonal rabbit anti-mouse TNF-receptor 1 antibody (1:500, Abcam, EngLand), respectively. After incubation with an horseradish peroxidase (HRP)-labeled goat IgG fraction to rabbit IgG Fc, the target protein was detected using a DAB kit (ZSGB-BIO, Beijing, China). The slides were then counterstained with hematoxylin and visualized under a microscope (Olympus, Tokyo, Japan).", "The slides were then counterstained with hematoxylin and visualized under a microscope (Olympus, Tokyo, Japan). Liver tissue and cells were homogenized in RIPA lysis buffer with phenyl methane sulfonyl fluoride (PMSF) protease inhibitor. Protein lysates were separated by SDS-PAGE, and western blotting was performed using a monoclonal mouse antihuman/mouse Fgl2 (1:750, Abnova), a monoclonal mouse antihuman HBP1 (1:100, Santa Cruz, USA), and a monoclonal rabbit anti-human/mouse β-actin (1:1,000, Cell Signaling Technology, USA).", "Protein lysates were separated by SDS-PAGE, and western blotting was performed using a monoclonal mouse antihuman/mouse Fgl2 (1:750, Abnova), a monoclonal mouse antihuman HBP1 (1:100, Santa Cruz, USA), and a monoclonal rabbit anti-human/mouse β-actin (1:1,000, Cell Signaling Technology, USA). Liver tissues were collected from MHV-3-infected BALB/cJ mice at 72 h, and total RNA was extracted using Trizol Reagent (Invitrogen, USA) and then reverse transcribed into cDNA by using ReverTra Ace qPCR RT kit (TOYOBO, Japan). The cDNA was then amplified by RT-PCR by using Dream Taq Green PCR Master Mix (2 ×) (Thermo Scientific, USA).", "The cDNA was then amplified by RT-PCR by using Dream Taq Green PCR Master Mix (2 ×) (Thermo Scientific, USA). Realtime quantitative PCR (qPCR) with SYBR Green Real-time PCR Master Mix (TOYOBO, Japan) was performed using a CFX96 real-time PCR detection system (Bio-Rad, USA) and mRNA levels were normalized with reference to those of the house keeping gene GAPDH. Primer sequences for qPCR amplification were as follows: mTNF-α forward, 5 ′ -TTT GAG ATC CAT GCC GTT GG-3 ′ ; mTNF-α reverse, 5 ′ -GCCA CCA CGC TCT TCT GT-3 ′ ; mIL-1β forward, 5 ′ -TGT AAT GAA AGA CGG CAC ACC-3 ′ ; mIL-1β reverse, 5 ′ -TCT TCT TTG GGT ATT GCT TGG-3 ′ .", "Primer sequences for qPCR amplification were as follows: mTNF-α forward, 5 ′ -TTT GAG ATC CAT GCC GTT GG-3 ′ ; mTNF-α reverse, 5 ′ -GCCA CCA CGC TCT TCT GT-3 ′ ; mIL-1β forward, 5 ′ -TGT AAT GAA AGA CGG CAC ACC-3 ′ ; mIL-1β reverse, 5 ′ -TCT TCT TTG GGT ATT GCT TGG-3 ′ . mFgl2 forward, 5 ′ -GCC AAA TGT GAG TCC CTG GAA-3 ′ ; mFgl2 reverse, 5 ′ -TTC CAC CCA AGA GCA CGT TTA AG-3 ′ ; hFgl2 forward 5 ′ -ACA GTT CAG GCT GGT GGT-3 ′ ; hFgl2 reverse, 5 ′ -GGC TTA AAG TGC TTG GGT-3 ′ ; HBP1 forward, 5 ′ -TGA AGC AGA AGC TGG GAGT-3 ′ ; HBP1 reverse,\n\nTHP-1 cells were treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma, USA) for 48 h to induce differentiation toward adherent macrophage-like cells as reported previously (24) . The CC10 group was supplemented with CC10 protein (150 ng/ml).", "The CC10 group was supplemented with CC10 protein (150 ng/ml). After 2 h of stimulation, IFN-γ (10 ng/ml) was added to these cells, which were then cultured for 12 h before they were collected for western blotting and real-time PCR studies. The Chinese hamster ovary (CHO) cells were cultured in 10 cm cell culture dishes with DMEM supplemented with 10% FBS until 80-90% confluence.", "The Chinese hamster ovary (CHO) cells were cultured in 10 cm cell culture dishes with DMEM supplemented with 10% FBS until 80-90% confluence. Next, 12 µg pcDNA3.1-hFgl2 (constructed in our lab) was mixed with 12 µg pcDNA3.1-hCC10 in serumfree DMEM. The mixture was then combined with Lipofectamine 2,000 (Invitrogen, USA) and mixed gently.", "The mixture was then combined with Lipofectamine 2,000 (Invitrogen, USA) and mixed gently. After incubation at 27 • C for 20 min, the solution was added to CHO cells and incubated at 37 • C in 5% CO 2 . Four to Six hour after transfection, the medium was removed and fresh medium containing 10% FBS was added.", "Four to Six hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, the cells were collected for co-immunoprecipitation analysis to evaluate the interaction of CC10 with Fgl2. Both HUVEC and THP-1 cells express fgl2.", "Both HUVEC and THP-1 cells express fgl2. However, in the transfection experiments, it is difficult to transfect the THP-1 cells with siRNA, so we use HUVEC instead of THP-1. Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in FIGURE 1 | CC10 protein increased survival rate and reduced liver damage in mice.", "Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in FIGURE 1 | CC10 protein increased survival rate and reduced liver damage in mice. (A) The survival rate of CC10 group is higher than the control group comprised of MHV-3-infected BALB/cJ mice treated with saline. CC10 protein (2 µg) or saline were injected into mice by tail vein.", "CC10 protein (2 µg) or saline were injected into mice by tail vein. BALB/cJ mice then received 100 PFU of MHV-3 intraperitoneally 24 h later to develop fulminant viral hepatitis. Then, CC10 protein (2 µg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later.", "Then, CC10 protein (2 µg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later. The survival rate was observed for 10 days (n = 24/group). Representative data from three independent experiments are shown. The survival curve was analyzed by using the Log-Rank Test.", "The survival curve was analyzed by using the Log-Rank Test. ***P < 0.001 compared with saline group. (B) Histopathology of liver tissues (H&E staining; original magnification, ×400, n = 5/group) at 72 h post-MHV-3 infection was evaluated in the two groups of MHV-3-infected BALB/cJ mice.", "(B) Histopathology of liver tissues (H&E staining; original magnification, ×400, n = 5/group) at 72 h post-MHV-3 infection was evaluated in the two groups of MHV-3-infected BALB/cJ mice. Livers were collected from saline-treated (a) and CC10-treated (b) BALB/cJ mice at 72 h after MHV-3 infection. Arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation.", "Arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation. (C) Effect of CC10 on serum ALT and AST levels (n = 6-8/group). Values represent means and standard error of three independent experiments performed in triplicate. **P < 0.01 compared with the saline group.", "**P < 0.01 compared with the saline group. six-well plates with DMEM supplemented with 10% FBS until 70-80% confluence. 50 pmol HBP1-siRNA was mixed with 125 µl serum-free DMEM. Two microliter Lipofectamine 2,000 was gently mixed with serum-free DMEM.", "Two microliter Lipofectamine 2,000 was gently mixed with serum-free DMEM. After incubation at 27 • C for 5 min, the solution was added to HUVECs and incubated at 37 • C. Four hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, cells were collected for real-time PCR and western blot analysis to evaluate the effects of HBP1 on Fgl2.", "At 48 h after transfection, cells were collected for real-time PCR and western blot analysis to evaluate the effects of HBP1 on Fgl2. At 24 h after transfection, the CC10 group was supplemented with the CC10 protein (150 ng/mL). After 4 h of stimulation, IFN-γ (10 ng/mL) was added to these cells.", "After 4 h of stimulation, IFN-γ (10 ng/mL) was added to these cells. These cells were then cultured for 24 h before they were harvested for real-time PCR studies to evaluate the effects of CC10 on Fgl2 by HBP1. Negative control was used as a control.", "Negative control was used as a control. To detect whether there was a potential interaction between CC10 protein and Fgl2, CHO cells were transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2 for 48 h. Cells transfected with empty plasmid pcDNA3.1 (mock) were used as negative controls for CC10 gene transfection. Immunoprecipitation and immunoblotting were performed by using Pierce Co-Immunoprecipitation Kit (Pierce, USA).", "Immunoprecipitation and immunoblotting were performed by using Pierce Co-Immunoprecipitation Kit (Pierce, USA). Total cell proteins were extracted as previously described (25) . The proteins were immunoprecipitated by mouse anti-human Fgl2 antibody (1:500, Abnova).", "The proteins were immunoprecipitated by mouse anti-human Fgl2 antibody (1:500, Abnova). For co-immunoprecipitation experiments, western blotting was performed using both rat anti-human uteroglobin/SCGB1A1 Antibody (1:750, R&D, USA) Frontiers in Immunology | and mouse anti-human Fgl2 antibody (1:500, Abnova). Control isotype rat IgG1 was used as a negative control for primary antibodies.", "Control isotype rat IgG1 was used as a negative control for primary antibodies. The human CC10 coding region gene, including a 389 bp sequence, was amplified from homogenized human turbinate tissue by RT-PCR. In this study, the sequences of PCR primers for CC10 were as follows: hCC10-forward, 5 ′ -CCC TCC ACC ATG AAA CTCG-3 ′ ; hCC10-reverse, 5 ′ -TGA GAT GCT TGT GGT TTA TTG AAG-3 ′ .", "In this study, the sequences of PCR primers for CC10 were as follows: hCC10-forward, 5 ′ -CCC TCC ACC ATG AAA CTCG-3 ′ ; hCC10-reverse, 5 ′ -TGA GAT GCT TGT GGT TTA TTG AAG-3 ′ . The PCR products were cloned into pEASY-T1 cloning vector (TransGEN, Beijing, China) and then subcloned into HindIII/XbaI site of pcDNA3.1 vector (Invitrogen, USA) to form eukaryotic expression plasmids pcDNA3.1-hCC10. Microarray analysis was used to screen changes in genome-wide gene expression patterns in THP-1 cells with or without CC10 protein.", "Microarray analysis was used to screen changes in genome-wide gene expression patterns in THP-1 cells with or without CC10 protein. The changes in over 47,000 human gene expression patterns were assessed using Affymetrix gene microarrays (Human Genome U133 Plus 2.0) (CapitalBio Co.,Ltd., Beijing, China). Three replicates were used for microarrays analysis.", "Three replicates were used for microarrays analysis. Data obtained from the experiments are expressed as means ± SEM. Survival curve comparisons were performed with the Log Rank test. Multiple group analyses for data were evaluated by one-way analyses of variance. Analyses of two group results were performed using Student's t-test to evaluate the statistical significance of differences.", "Analyses of two group results were performed using Student's t-test to evaluate the statistical significance of differences. Values of P < 0.05 indicated significance. To establish an animal model of mouse FH, MHV-3 was injected intraperitoneally to BALB/cJ mice (24 mice/group).", "To establish an animal model of mouse FH, MHV-3 was injected intraperitoneally to BALB/cJ mice (24 mice/group). To further study the role of CC10 in FH, recombinant mouse CC10 protein (2 µg/mouse) or saline was administrated into the tail vein 24 h prior to MHV-3 infection. The same dose of CC10 protein or saline was then administered 24 h later.", "The same dose of CC10 protein or saline was then administered 24 h later. The survival rate of the CC10 and saline groups was observed for 10 days. The results showed that mice in the two groups began to die at 48 h after injection of MHV-3 and exhibited symptoms of horripilation, slow activity, and reduced food consumption.", "The results showed that mice in the two groups began to die at 48 h after injection of MHV-3 and exhibited symptoms of horripilation, slow activity, and reduced food consumption. In the CC10 group 24 mice were alive on day 3 after infection, 4 mice alive on day 4, and 3 of 24 (12.5%) mice recovered from fulminant viral hepatitis. At the same time, in saline treated group, there were 5 mice alive on day 3, 1 mice alive on day 4 after infection, and no mice survived to day 5.", "At the same time, in saline treated group, there were 5 mice alive on day 3, 1 mice alive on day 4 after infection, and no mice survived to day 5. That is to say, the mice in the saline group died within 3 or 4 days. Three of 24 (12.5%) mice of the CC10 group recovered from fulminant viral hepatitis ( Figure 1A) .", "Three of 24 (12.5%) mice of the CC10 group recovered from fulminant viral hepatitis ( Figure 1A) . To better understand the mechanisms underlying the biological effects of the CC10 protein, liver function (ALT and AST levels in serum) and liver histology in mice of MHV-3-infected was performed. Liver tissues were harvested 72 h following MHV-3 infection, and liver histology was detected by H&E staining.", "Liver tissues were harvested 72 h following MHV-3 infection, and liver histology was detected by H&E staining. These results showed that there was substantial inflammatory cell infiltration and widespread necrosis of hepatocytes in the liver tissue of the saline group mice (Figure 1Ba ). There were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the CC10 group 72 h after MHV-3 infection (Figure 1Bb) .", "There were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the CC10 group 72 h after MHV-3 infection (Figure 1Bb) . Serum ALT and AST levels in mice were observed 72 h after MHV-3 infection. The results showed that serum ALT and AST levels in the saline group reached a peak 72 h after MHV-3 infection, but there was no significant increase in the CC10 group compared to the levels in the control group (P < 0.01, Figure 1C) .", "The results showed that serum ALT and AST levels in the saline group reached a peak 72 h after MHV-3 infection, but there was no significant increase in the CC10 group compared to the levels in the control group (P < 0.01, Figure 1C) . These results suggested that CC10 protein has a role in protection against MHV-3-induced liver injury in mice. To further elucidate the mechanisms of reduced liver injury following CC10 protein injection, we investigated the cytokines TNF-α and IL-1β expression.", "To further elucidate the mechanisms of reduced liver injury following CC10 protein injection, we investigated the cytokines TNF-α and IL-1β expression. Because these two cytokines play a crucial role in the liver damage of FH. They are characterized by an increase in apoptosis.", "They are characterized by an increase in apoptosis. Levels of TNF-α and IL-1β in liver tissues were markedly reduced in the CC10 group (as shown in Figure 2A) . Hepatic apoptosis (Figure 2B ) was significantly reduced in the CC10 group.", "Hepatic apoptosis (Figure 2B ) was significantly reduced in the CC10 group. We and collaborators have a long standing interest in studying the role of fgl2 in viral hepatitis. Fgl2 has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports.", "Fgl2 has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports. We have provided liver pathology figures and liver function for MHV-3 infected mice with a fgl2 gene knockout as shown in Supplementary Figure 1 . The data was comparable with previous reports from our center and collaborators.", "The data was comparable with previous reports from our center and collaborators. From this current study we shown that CC10 plays a protective role in liver damage.To study the related molecules of CC10 in MHV-3-induced FH mice, we evaluated whether there was crosstalk between Fgl2 and CC10. We found that the expression of Fgl2 in the liver of mice was reduced 72 h after MHV-3 infection and treatment with CC10 protein (Figures 3A,B) .", "We found that the expression of Fgl2 in the liver of mice was reduced 72 h after MHV-3 infection and treatment with CC10 protein (Figures 3A,B) . Furthermore, fibrin deposition, an indicator of liver injury associated with Fgl2 expression in FH, was also decreased in the livers of CC10-treated mice compared to that in controls (Figure 3C ). This indicates that CC10 treatment reduced liver injury after viral infection by inhibiting Fgl2 expression.", "This indicates that CC10 treatment reduced liver injury after viral infection by inhibiting Fgl2 expression. We examined the effect of increasing doses of CC10 protein (0, 50, 150, and 300 ng/mL) on IFN-γ-induced Fgl2 expression in THP-1 cells. CC10 treatment showed a 10.1% decrease in THP-1 cells compared to that in control after stimulation with 10 ng/mL IFN-γ for 12 h. CC10 protein inhibited Fgl2 expression between doses of 0 ng/mL and 300 ng/mL (Figure 4A ).", "CC10 treatment showed a 10.1% decrease in THP-1 cells compared to that in control after stimulation with 10 ng/mL IFN-γ for 12 h. CC10 protein inhibited Fgl2 expression between doses of 0 ng/mL and 300 ng/mL (Figure 4A ). In particular, 150 ng/mL CC10 protein had the strongest inhibitory effect on Fgl2 expression among the doses, and we chose this dose for the following experiments. We explored the effect of different time points of stimulation with a concentration of 150 ng/mL CC10 protein.", "We explored the effect of different time points of stimulation with a concentration of 150 ng/mL CC10 protein. After stimulation with CC10 protein for 6, 12, and 24 h compared to the PBS control, the strongest inhibitory effect on Fgl2 expression was noted at 12 h; hence, we chose this time point for the following studies ( Figure 4B ). An increasing number of studies suggest that macrophages are the primary source of Fgl2.", "An increasing number of studies suggest that macrophages are the primary source of Fgl2. In order to ascertain that CC10 has a direct effect on macrophages, we treated THP-1 cells with recombinant CC10 and assessed the expression of Fgl2. Unlike in controls, IFN-γ induced a significant increase in Fgl2 expression.", "Unlike in controls, IFN-γ induced a significant increase in Fgl2 expression. This effect was attenuated when cells were treated with CC10 protein (Figures 4C,D) , revealing that CC10 directly reduces the levels of Fgl2 in macrophages. To further explore the possibility that CC10 protein directly acts on macrophages, we infected murine PEMs with MHV-3 in the presence of recombinant CC10 and determined Fgl2 expression.", "To further explore the possibility that CC10 protein directly acts on macrophages, we infected murine PEMs with MHV-3 in the presence of recombinant CC10 and determined Fgl2 expression. Compared to levels in the controls, MHV-3infected macrophages exhibited a significant increase in Fgl2 production, and this effect was abolished by using CC10 protein (Figures 5A,B) , indicating that CC10 directly modulates Fgl2 production in macrophages. In order to determine genes that were downregulated after stimulation by CC10 protein, we used DNA microarray analysis to screen for differentially expressed genes.", "In order to determine genes that were downregulated after stimulation by CC10 protein, we used DNA microarray analysis to screen for differentially expressed genes. THP-1 cells were cultured and PMA was added to induce differentiation into macrophages. The production of Fgl2 was stimulated by IFNγ.", "The production of Fgl2 was stimulated by IFNγ. The experimental group was treated with CC10 protein for microarray detection of differentially expressed genes. The results showed that the most obviously downregulated genes were UBE2W, HECTD1, MIR612, ATRX, SOX4, HBP1, and Fgl2 (Supplementary Table 1) .", "The results showed that the most obviously downregulated genes were UBE2W, HECTD1, MIR612, ATRX, SOX4, HBP1, and Fgl2 (Supplementary Table 1) . And then these genes were tested by qPCR. However, UBE2W, HECTD1, MIR612, ATRX, and SOX4 was not differentially expressed by qPCR, while HBP1 and fgl2 were still down-regulated genes.", "However, UBE2W, HECTD1, MIR612, ATRX, and SOX4 was not differentially expressed by qPCR, while HBP1 and fgl2 were still down-regulated genes. DNA microarray analysis identified HBP1 as a down-regulated gene involved in the pathological processes of the regulation of CC10. Recently, very limited studies have explored the role of HBP1 in FH.", "Recently, very limited studies have explored the role of HBP1 in FH. Nevertheless, the mechanistic functions of HBP1 in FH remain largely unexplored. Therefore, we selected this gene for further study.", "Therefore, we selected this gene for further study. qPCR analysis confirmed that mRNA levels of HBP1 were significantly decreased in THP-1 cells after CC10\n\nprotein stimulation compared to that in the PBS control group (Figure 6A ). We knocked down HBP1 using HBP1-siRNA. Then, transfection of HBP1-SiRNA into HUVECs was detected by qPCR and western-blotting methods.", "Then, transfection of HBP1-SiRNA into HUVECs was detected by qPCR and western-blotting methods. As expected, HBP1 knockdown led to significantly decreased expression of HBP1 (Figures 6B,C) . Furthermore, HBP1 knockdown impaired expression of Fgl2 (Figure 6D ), suggesting that HBP1 was able to activate Fgl2. HBP1-SiRNA was used to transfect HUVECs.", "HBP1-SiRNA was used to transfect HUVECs. Then, IFN-γ was added to induce the expression of Fgl2 followed by stimulation with CC10 protein (150 ng/ml) after 2 h. Finally, we explored the expression of Fgl2 by qPCR. The results showed that HBP1-SiRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in HUVECs (Figure 7) .", "The results showed that HBP1-SiRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in HUVECs (Figure 7) . That is to say, CC10 could suppress Fgl2 expression in macrophages. Such an effect may be mediated by the transcription factor HBP1. It is well-known that CC10 protein can suppress the immune response.", "It is well-known that CC10 protein can suppress the immune response. In animal models of allergic diseases of the respiratory tract, most of evidences confirm this inhibition (26) . Its function in FH has not been investigated yet.", "Its function in FH has not been investigated yet. Here, we used a murine FH model established by MHV-3 infection to explore the effects of CC10 in this disease process. To determine the role of CC10 in the pathogenesis of FH, CC10 protein was injected into a mouse FH model established by MHV-3 infection.", "To determine the role of CC10 in the pathogenesis of FH, CC10 protein was injected into a mouse FH model established by MHV-3 infection. MHV-3-induced liver injury in CC10-treated mice occurred rarely and the areas of lesions were much fewer than those in saline-treated control mice. In summary, these results suggested that CC10 could reduce pathological liver damage in this FH model together with lower mortality rates followed by MHV-3 infection.", "In summary, these results suggested that CC10 could reduce pathological liver damage in this FH model together with lower mortality rates followed by MHV-3 infection. MHV-3 induced fulminant viral hepatitis progresses rapidly and infected mice die within 3-5 days. Previous studies suggested fgl2 played a vital role in this process with a 15-40% increase of survival when fgl2 was deleted (12, 15, 27, 28) .", "Previous studies suggested fgl2 played a vital role in this process with a 15-40% increase of survival when fgl2 was deleted (12, 15, 27, 28) . Multiple inflammatory factors or mediators including TNF-α and IFN-γ, IL-1β and C5aR have been demonstrated to promote FH progression with significant discrepancies between liver damage and survival rate (29) (30) (31) (32) , which is accordant with our observation that CC10 substantially alleviated liver injury though survival rate improved mildly. The survival rate based on hours may be more accurate to examine the effect of CC10 on FH.", "The survival rate based on hours may be more accurate to examine the effect of CC10 on FH. It is speculated that fgl2 can mediate lethality in MHV-3-induced FH. This is due to the fact that fgl2 induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity (15) .", "This is due to the fact that fgl2 induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity (15) . To determine whether the tissue necrosis was mediated by Fgl2 in CC10-treated mice following infection, Fgl2 expression was observed. Results suggested that the expression of Fgl2 was significantly increased in MHV-3-induced FH mice and CC10 treatment significantly reduced the production of Fgl2 in the infected liver and serum.", "Results suggested that the expression of Fgl2 was significantly increased in MHV-3-induced FH mice and CC10 treatment significantly reduced the production of Fgl2 in the infected liver and serum. In addition, decreased fibrinogen deposition was also observed in the livers of CC10-treated mice. Therefore, our research results strongly clarify that the lower mortality of CC10-treated mice after MHV-3 infection is due to the lower levels of Fgl2 and decreased fibrinogen deposition.", "Therefore, our research results strongly clarify that the lower mortality of CC10-treated mice after MHV-3 infection is due to the lower levels of Fgl2 and decreased fibrinogen deposition. Indeed, it has been reported that Fgl2 is expressed on macrophages, and the expression of Fgl2 is believed to be induced by IFN-γ and TNF-α (22) . Cultured THP-1 cells activated by IFN-γ or IL-2 have been demonstrated, with induction of Fgl2 expression and enhanced activation of human prothrombin (23) .", "Cultured THP-1 cells activated by IFN-γ or IL-2 have been demonstrated, with induction of Fgl2 expression and enhanced activation of human prothrombin (23) . Therefore, in this study, we explored this cell line to investigate the modulation of CC10 on Fgl2. Surprisingly, we found that CC10 directly inhibited IFN-γ-induced Fgl2 expression in THP-1 cells.", "Surprisingly, we found that CC10 directly inhibited IFN-γ-induced Fgl2 expression in THP-1 cells. As we know, IFN-γ has proved to be the main cytokine that leads to the development and progression of FH. Also, it was shown that IFN-γ might exert its own proinflammatory biological function through enhancing Fgl2 expression.", "Also, it was shown that IFN-γ might exert its own proinflammatory biological function through enhancing Fgl2 expression. Therefore, in our study, CC10 might counter the effect of IFN-γ in the setting of FH, which substantiates its role in FH. These results demonstrated that CC10 regulates the expression of Fgl2 in macrophages.", "These results demonstrated that CC10 regulates the expression of Fgl2 in macrophages. In the current study, we used co-immunoprecipitation to analyze binding between CC10 and Fgl2. In this study, we investigated possible protein-protein interactions between CC10 and Fgl2 in vitro. The Chinese hamster ovary (CHO) cells transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2.", "The Chinese hamster ovary (CHO) cells transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2. Cellular proteins were immunoprecipitated with anti-CC10 antibody or anti-Fgl2 antibody. Immunoblotting was performed with anti-Fgl2 and anti-CC10 antibodies.", "Immunoblotting was performed with anti-Fgl2 and anti-CC10 antibodies. Immunoprecipitation of protein extracts from pcDNA 3.1-CC10 and pcDNA3.1-Fgl2 co-transfected CHO cells with anti-Fgl2 or anti-CC10 antibody followed by western blotting with Fgl2 and CC10 antibodies indicated that CC10 did not co-immunoprecipitate with Fgl2, showing that there is no direct relationship between CC10 and Fgl2 (data not shown). The results showed that CC10 has no direct interaction with Fgl2.", "The results showed that CC10 has no direct interaction with Fgl2. From our previous study the gene of fgl2 contributed profoundly in MHV-3 induced fulminant hepatitis and is extensively expressed in macrophages and endothelium (12, 33) . Our microarray indicated a CC10 down-regulated fgl2 expression and this is further confirmed by qPCR and Western blotting in vivo (peritoneal macrophages) and in vitro (THP-1, macrophage cell line).", "Our microarray indicated a CC10 down-regulated fgl2 expression and this is further confirmed by qPCR and Western blotting in vivo (peritoneal macrophages) and in vitro (THP-1, macrophage cell line). Therefore, it is reasonable to focus on macrophages to display the effect of CC10 on fgl2 expression and eventually mice survival. We entirely agree there may be other possibilities for a protective effect of CC10 to contribute to the disease process.", "We entirely agree there may be other possibilities for a protective effect of CC10 to contribute to the disease process. This is worth further studies. The potential receptor of CC10 has not been revealed yet. Our previous study have demonstrated that CC10 have effect of dendritic cells in allergic rhinitis (34) .", "Our previous study have demonstrated that CC10 have effect of dendritic cells in allergic rhinitis (34) . In this research, we evaluated the effect of CC10 on macrophages functions and found Fgl2 was substantially down-regulated upon CC10 treatment, therefore, we speculate that potential CC10 receptor may be also expressed on macrophages. The potential target of CC10 on other immune cells cannot be excluded.", "The potential target of CC10 on other immune cells cannot be excluded. DNA microarray analysis is one of the most powerful approaches for the potential identification of unexpected genes involved in pathogenic processes. By using this approach, HMGbox transcription factor 1 (HBP1) was found to be one of the most downregulated genes after CC10 treatment of THP-1 cells.", "By using this approach, HMGbox transcription factor 1 (HBP1) was found to be one of the most downregulated genes after CC10 treatment of THP-1 cells. HBP1 is a well-described transcriptional repressor that modulates expression of genes involved in cell cycle progression. In a recent study, it was found that HBP1 is a direct target of miR-21 and confirmed that HBP1 modulates the inhibitory function of miR-21-ASO in hepatosteatosis and carcinogenesis simultaneously (23) .", "In a recent study, it was found that HBP1 is a direct target of miR-21 and confirmed that HBP1 modulates the inhibitory function of miR-21-ASO in hepatosteatosis and carcinogenesis simultaneously (23) . HBP1 is an endogenous inhibitor of the Wnt signaling pathway in both normal and cancer cells. The tumor suppressor role of HBP1 has been reported in some malignancies, such as oral cancer and glioma (35) .", "The tumor suppressor role of HBP1 has been reported in some malignancies, such as oral cancer and glioma (35) . However, an association between HBP1 and Fgl2 has not been investigated yet. The current study clearly demonstrated that CC10 protects against MHV-3 induced FH via suppression of Fgl2 expression. Such effects might be mediated by HBP1.", "Such effects might be mediated by HBP1. However, the functional status of HBP1 in the CC10 pathway requires further research, and such studies are conducting in our laboratory. In conclusion, we demonstrated that CC10 could limit the immunopathological damage in MHV-3-induced FH mice.", "In conclusion, we demonstrated that CC10 could limit the immunopathological damage in MHV-3-induced FH mice. Our results suggest that enhancing CC10 expression by an immunotherapeutic approach might be an effective treatment for FH. HY performed all the described experiments and wrote the manuscript. YL assisted with some experiments, analyzed experimental results, and edited the manuscript.", "YL assisted with some experiments, analyzed experimental results, and edited the manuscript. HW analyzed experimental results. XW reviewed and edited the manuscript. JH, WY, DX, XL, GS, and QN provided experimental help and design." ]

[ "Background: Fulminant hepatitis (FH) is a serious threat to human life, accompanied by massive and rapid necroinflammation. Kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for FH. Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection.", "Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection. Clara cell 10 kDa (CC10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. However, its mechanisms of action and pathogenic roles in other disease are still unclear.", "However, its mechanisms of action and pathogenic roles in other disease are still unclear. In this study, we aimed to determine the role of CC10 in FH and the regulation of Fgl2 by CC10. Methods: A mouse FH model was established by peritoneal injection of MHV-3.", "Methods: A mouse FH model was established by peritoneal injection of MHV-3. The mice received CC10 protein through tail vein injection before viral infection. Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined.", "Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. The regulatory effect of CC10 on Fgl2 expression was investigated using THP-1 cells and mouse peritoneal macrophages in vitro. Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group.", "Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group. Meanwhile, the levels of ALT and AST in serum were significantly decreased and liver damage was reduced. Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein.", "Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein. In vitro, CC10 was found to significantly inhibit the expression of Fgl2 in IFN-γ-treated THP-1 cells and MHV-3-infected mouse peritoneal macrophages by western blot and real-time PCR. However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation.", "However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation. Microarray investigations suggested that HMG-box transcription factor 1 (HBP1) was significantly low in CC10-treated and IFN-γ-primed THP-1 cells. HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs).", "HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs). Conclusion:CC10 protects against MHV-3-induced FH via suppression of Fgl2 expression in macrophages. Such effects may be mediated by the transcription factor HBP1.", "Such effects may be mediated by the transcription factor HBP1. Text: Fulminant hepatitis (FH) is a serious life-threatening disease characterized by massive hepatocyte necrosis, severe liver damage, and high mortality. The underlying mechanisms and the pathogenesis of FH are not clear.", "The underlying mechanisms and the pathogenesis of FH are not clear. However, accumulating evidence suggests that, regardless of the pathogenesis of FH, the host's inflammatory responses contribute to liver microcirculatory disorders and injuries. Accordingly, It has been shown that immune cell activation and inflammatory cytokines play an important role in FH (1) .", "Accordingly, It has been shown that immune cell activation and inflammatory cytokines play an important role in FH (1) . In recent years, our laboratory has conducted extensive research on the pathogenesis of FH and found that immune cells play a key role in it. Kupffer cells, natural killer (NK) cells (2, 3) , cytotoxic T-lymphocytes (CTLs), and double negative T-cells (DNT) (4) (5) (6) in liver and the cytokines that are produced by these cells cause liver damage.", "Kupffer cells, natural killer (NK) cells (2, 3) , cytotoxic T-lymphocytes (CTLs), and double negative T-cells (DNT) (4) (5) (6) in liver and the cytokines that are produced by these cells cause liver damage. Prothrombinase Fgl2 belongs to the fibrinogen superfamily and is produced by activated macrophages or endothelial cells, transforming prothrombin directly into thrombin, so as to quickly initiate the process of coagulation. This promotes the conversion of fibrinogen into fibrin, resulting in thrombosis (7) (8) (9) (10) (11) (12) .", "This promotes the conversion of fibrinogen into fibrin, resulting in thrombosis (7) (8) (9) (10) (11) (12) . Our study found that Fgl2 was highly expressed in peripheral blood mononuclear cells (PBMCs) and in liver tissue of humans or mice with severe viral hepatitis, and was positively related to the severity of the disease (13, 14) . Gene therapy targeting Fgl2 silencing showed that the survival rate of fulminant hepatitis mice increased from 0 to 33.3% (15) .", "Gene therapy targeting Fgl2 silencing showed that the survival rate of fulminant hepatitis mice increased from 0 to 33.3% (15) . Thus far, the discovery and related research involving Fgl2 have provided new insights into the molecular mechanism of hepatocyte necrosis in FH. In view of the important role of Fgl2 in severe viral hepatitis, investigations concerning the regulation of Fgl2 will be beneficial in the search for new strategies for treatment of severe hepatitis.", "In view of the important role of Fgl2 in severe viral hepatitis, investigations concerning the regulation of Fgl2 will be beneficial in the search for new strategies for treatment of severe hepatitis. Clara cell 10 kDa protein (CC10), also considered to be uteroglobin, Clara cell secretory protein, is one of members of secretoglobin superfamily. Expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world (16) .", "Expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world (16) . CC10 has immunomodulatory and anti-inflammatory effects. Compared to wild-type mice, CC10-knockout mice exhibited excessive airway inflammation Abbreviations: FH, fulminant hepatitis; MHV-3, murine hepatitis virus strain 3; Fgl2, Fibrinogen-like protein 2; CC10, Clara cell 10 KDa protein; ALF, acute liver failure; PFU, plaque-forming units; PBS, phosphate-buffered saline; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCA, pro-coagulant activity; HRP, horseradish peroxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.", "Compared to wild-type mice, CC10-knockout mice exhibited excessive airway inflammation Abbreviations: FH, fulminant hepatitis; MHV-3, murine hepatitis virus strain 3; Fgl2, Fibrinogen-like protein 2; CC10, Clara cell 10 KDa protein; ALF, acute liver failure; PFU, plaque-forming units; PBS, phosphate-buffered saline; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCA, pro-coagulant activity; HRP, horseradish peroxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. caused by allergic reaction and bacterial and viral infections (17) . Reduced levels of CC10 are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis (18) (19) (20) (21) .", "Reduced levels of CC10 are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis (18) (19) (20) (21) . Previous studies and published articles show that CC10 protein can not only inhibit Th17 cell responses by inhibiting expression of related molecules of dendritic cells and cytokines in mice with allergic rhinitis, but also can inhibit chitosan-3 like protein 1 (22, 23) . Moreover, CC10 inhibits the expression of an important immune regulator, osteopontin (OPN), in models of allergic rhinitis (21) .", "Moreover, CC10 inhibits the expression of an important immune regulator, osteopontin (OPN), in models of allergic rhinitis (21) . In this study, we investigated the role of CC10 in hepatitis virus strain 3 (MHV-3)-induced FH in mice and explored whether CC10 protein could regulate Fgl2 in the disease process. Female BALB/cJ mice (Shanghai Shilaike Animal Seed Center, Shanghai, China), 6-8 weeks of age, with a body weight of 18.0-20.0 g, were kept in Tongji Hospital with food and water.", "Female BALB/cJ mice (Shanghai Shilaike Animal Seed Center, Shanghai, China), 6-8 weeks of age, with a body weight of 18.0-20.0 g, were kept in Tongji Hospital with food and water. Mice were divided into two groups: CC10 group (experimental group) and phosphate-buffered saline (PBS) group (control group). This study was carried out in accordance with the recommendations of the guidelines of the National Institutes of Health and the Animal Experiment Committee of Tongji hospital.", "This study was carried out in accordance with the recommendations of the guidelines of the National Institutes of Health and the Animal Experiment Committee of Tongji hospital. This study was reviewed and approved by the Animal Experiment Committee of Tongji hospital. The human monocyte cell line THP-1 was purchased from the Cell Institute of the Chinese Academy of Sciences (Shanghai, China).", "The human monocyte cell line THP-1 was purchased from the Cell Institute of the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from the Biology Treasure Center of Wuhan University, China. The Chinese hamster ovary (CHO) cell line was acquired from the typical culture preservation commission cell bank, the Chinese Academy of Sciences (Shanghai, China).", "The Chinese hamster ovary (CHO) cell line was acquired from the typical culture preservation commission cell bank, the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) and CHO cells were cultured in Dulbecco's modified Eagle's medium (DMEM), and THP-1 cells were maintained in RPMI 1,640 containing 10% heat inactivated fetal bovine serum (FBS, Gibco Life Technologies, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin and cultured at 37 • C, 50 mL/L CO 2 and 95% humidity. Peritoneal exudative macrophages (PEMs) were obtained from BALB/cJ mice.", "Peritoneal exudative macrophages (PEMs) were obtained from BALB/cJ mice. Cells were resuspended in RPMI 1,640 supplemented with 10% FBS at 1-2 × 10 6 cells/mL in a 6-well plate and incubated for 4 h. They were then washed with RPMI 1640 medium and non-adherent cells discarded. The adherent cells were macrophages and were incubated for a further 12 h. Peritoneal exudative macrophages (PEMs) were divided into two groups.", "The adherent cells were macrophages and were incubated for a further 12 h. Peritoneal exudative macrophages (PEMs) were divided into two groups. One group was supplemented with CC10 protein (150 ng/mL) and in the other group, PBS was added. After 2 h of stimulation, 1,000 plaque forming units (PFUs) of MHV-3 was added to the cells, which were then cultured for 4 h. Peritoneal exudative macrophages (PEMs) were harvested and lysed for real-time PCR and western blotting analysis.", "After 2 h of stimulation, 1,000 plaque forming units (PFUs) of MHV-3 was added to the cells, which were then cultured for 4 h. Peritoneal exudative macrophages (PEMs) were harvested and lysed for real-time PCR and western blotting analysis. Cell apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method with a TUNEL apoptosis detection kit (Roche, Switzerland). Briefly, 5 µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase K for 30 min at 37 • C. After stopping the proteinase K digestion reaction with PBS, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dUTP at a ratio of 2:29, respectively), for 2 h at 37 • C in an immunohistochemistry wet box.", "Briefly, 5 µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase K for 30 min at 37 • C. After stopping the proteinase K digestion reaction with PBS, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dUTP at a ratio of 2:29, respectively), for 2 h at 37 • C in an immunohistochemistry wet box. Following washing and blocking, each section was supplemented with reagent (converter-POD) to cover the tissues and incubated for 30 min at 37 • C in a wet box. Then, the liver tissue sections were washed with PBS, and colored with diaminobenzidine (DAB) subsequently.", "Then, the liver tissue sections were washed with PBS, and colored with diaminobenzidine (DAB) subsequently. Hepatocytes with nucleus stained brownish yellow were considered to be apoptotic cells. The expression of Fgl2 on THP-1 cells was measured by flow cytometry (BD FACS Canto II, USA).", "The expression of Fgl2 on THP-1 cells was measured by flow cytometry (BD FACS Canto II, USA). Briefly, cells (2 × 10 5 per tube) were incubated with Human TruStrain FcX (Fc Receptor Blocking solution, BioLegend, USA) for 10 min at room temperature and then incubated in the dark with mouse anti-Fgl2 antibody (1:100, Abnova,) or normal goat serum (an isotype control) at 4 • C for 40 min. Cells were washed with PBS and incubated in the dark with PE-conjugated goat anti-mouse IgG antibody (1:50, BioLegend, USA) at 4 • C for 30 min.", "Cells were washed with PBS and incubated in the dark with PE-conjugated goat anti-mouse IgG antibody (1:50, BioLegend, USA) at 4 • C for 30 min. Cells were then washed with PBS and resuspended in 300 µL PBS for study. Liver slices were fixed in 4% paraformaldehyde and then embedded in paraffin.", "Liver slices were fixed in 4% paraformaldehyde and then embedded in paraffin. Immunohistochemistry of liver tissues was performed using SP-9001 SPlink Detection Kits (Biotin-Streptavidin HRP Detection Systems) (ZSGB-BIO, Beijing, China) according to the manufacturer's instructions. For immunohistochemistry staining, the expression of Fgl2, fibrinogen, Fas and TNF-receptor 1 in mouse liver tissues was detected with polyclonal rabbit anti-mouse Fgl2 antibody (1:100, Proteintech, USA), polyclonal rabbit anti-mouse fibrinogen antibody (1:1,000, Abcam, EngLand), polyclonal rabbit antimouse Fas antibody (1:50, Abcam, EngLand), and polyclonal rabbit anti-mouse TNF-receptor 1 antibody (1:500, Abcam, EngLand), respectively.", "For immunohistochemistry staining, the expression of Fgl2, fibrinogen, Fas and TNF-receptor 1 in mouse liver tissues was detected with polyclonal rabbit anti-mouse Fgl2 antibody (1:100, Proteintech, USA), polyclonal rabbit anti-mouse fibrinogen antibody (1:1,000, Abcam, EngLand), polyclonal rabbit antimouse Fas antibody (1:50, Abcam, EngLand), and polyclonal rabbit anti-mouse TNF-receptor 1 antibody (1:500, Abcam, EngLand), respectively. After incubation with an horseradish peroxidase (HRP)-labeled goat IgG fraction to rabbit IgG Fc, the target protein was detected using a DAB kit (ZSGB-BIO, Beijing, China). The slides were then counterstained with hematoxylin and visualized under a microscope (Olympus, Tokyo, Japan).", "The slides were then counterstained with hematoxylin and visualized under a microscope (Olympus, Tokyo, Japan). Liver tissue and cells were homogenized in RIPA lysis buffer with phenyl methane sulfonyl fluoride (PMSF) protease inhibitor. Protein lysates were separated by SDS-PAGE, and western blotting was performed using a monoclonal mouse antihuman/mouse Fgl2 (1:750, Abnova), a monoclonal mouse antihuman HBP1 (1:100, Santa Cruz, USA), and a monoclonal rabbit anti-human/mouse β-actin (1:1,000, Cell Signaling Technology, USA).", "Protein lysates were separated by SDS-PAGE, and western blotting was performed using a monoclonal mouse antihuman/mouse Fgl2 (1:750, Abnova), a monoclonal mouse antihuman HBP1 (1:100, Santa Cruz, USA), and a monoclonal rabbit anti-human/mouse β-actin (1:1,000, Cell Signaling Technology, USA). Liver tissues were collected from MHV-3-infected BALB/cJ mice at 72 h, and total RNA was extracted using Trizol Reagent (Invitrogen, USA) and then reverse transcribed into cDNA by using ReverTra Ace qPCR RT kit (TOYOBO, Japan). The cDNA was then amplified by RT-PCR by using Dream Taq Green PCR Master Mix (2 ×) (Thermo Scientific, USA).", "The cDNA was then amplified by RT-PCR by using Dream Taq Green PCR Master Mix (2 ×) (Thermo Scientific, USA). Realtime quantitative PCR (qPCR) with SYBR Green Real-time PCR Master Mix (TOYOBO, Japan) was performed using a CFX96 real-time PCR detection system (Bio-Rad, USA) and mRNA levels were normalized with reference to those of the house keeping gene GAPDH. Primer sequences for qPCR amplification were as follows: mTNF-α forward, 5 ′ -TTT GAG ATC CAT GCC GTT GG-3 ′ ; mTNF-α reverse, 5 ′ -GCCA CCA CGC TCT TCT GT-3 ′ ; mIL-1β forward, 5 ′ -TGT AAT GAA AGA CGG CAC ACC-3 ′ ; mIL-1β reverse, 5 ′ -TCT TCT TTG GGT ATT GCT TGG-3 ′ .", "Primer sequences for qPCR amplification were as follows: mTNF-α forward, 5 ′ -TTT GAG ATC CAT GCC GTT GG-3 ′ ; mTNF-α reverse, 5 ′ -GCCA CCA CGC TCT TCT GT-3 ′ ; mIL-1β forward, 5 ′ -TGT AAT GAA AGA CGG CAC ACC-3 ′ ; mIL-1β reverse, 5 ′ -TCT TCT TTG GGT ATT GCT TGG-3 ′ . mFgl2 forward, 5 ′ -GCC AAA TGT GAG TCC CTG GAA-3 ′ ; mFgl2 reverse, 5 ′ -TTC CAC CCA AGA GCA CGT TTA AG-3 ′ ; hFgl2 forward 5 ′ -ACA GTT CAG GCT GGT GGT-3 ′ ; hFgl2 reverse, 5 ′ -GGC TTA AAG TGC TTG GGT-3 ′ ; HBP1 forward, 5 ′ -TGA AGC AGA AGC TGG GAGT-3 ′ ; HBP1 reverse,\n\nTHP-1 cells were treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma, USA) for 48 h to induce differentiation toward adherent macrophage-like cells as reported previously (24) . The CC10 group was supplemented with CC10 protein (150 ng/ml).", "The CC10 group was supplemented with CC10 protein (150 ng/ml). After 2 h of stimulation, IFN-γ (10 ng/ml) was added to these cells, which were then cultured for 12 h before they were collected for western blotting and real-time PCR studies. The Chinese hamster ovary (CHO) cells were cultured in 10 cm cell culture dishes with DMEM supplemented with 10% FBS until 80-90% confluence.", "The Chinese hamster ovary (CHO) cells were cultured in 10 cm cell culture dishes with DMEM supplemented with 10% FBS until 80-90% confluence. Next, 12 µg pcDNA3.1-hFgl2 (constructed in our lab) was mixed with 12 µg pcDNA3.1-hCC10 in serumfree DMEM. The mixture was then combined with Lipofectamine 2,000 (Invitrogen, USA) and mixed gently.", "The mixture was then combined with Lipofectamine 2,000 (Invitrogen, USA) and mixed gently. After incubation at 27 • C for 20 min, the solution was added to CHO cells and incubated at 37 • C in 5% CO 2 . Four to Six hour after transfection, the medium was removed and fresh medium containing 10% FBS was added.", "Four to Six hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, the cells were collected for co-immunoprecipitation analysis to evaluate the interaction of CC10 with Fgl2. Both HUVEC and THP-1 cells express fgl2.", "Both HUVEC and THP-1 cells express fgl2. However, in the transfection experiments, it is difficult to transfect the THP-1 cells with siRNA, so we use HUVEC instead of THP-1. Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in FIGURE 1 | CC10 protein increased survival rate and reduced liver damage in mice.", "Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in FIGURE 1 | CC10 protein increased survival rate and reduced liver damage in mice. (A) The survival rate of CC10 group is higher than the control group comprised of MHV-3-infected BALB/cJ mice treated with saline. CC10 protein (2 µg) or saline were injected into mice by tail vein.", "CC10 protein (2 µg) or saline were injected into mice by tail vein. BALB/cJ mice then received 100 PFU of MHV-3 intraperitoneally 24 h later to develop fulminant viral hepatitis. Then, CC10 protein (2 µg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later.", "Then, CC10 protein (2 µg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later. The survival rate was observed for 10 days (n = 24/group). Representative data from three independent experiments are shown. The survival curve was analyzed by using the Log-Rank Test.", "The survival curve was analyzed by using the Log-Rank Test. ***P < 0.001 compared with saline group. (B) Histopathology of liver tissues (H&E staining; original magnification, ×400, n = 5/group) at 72 h post-MHV-3 infection was evaluated in the two groups of MHV-3-infected BALB/cJ mice.", "(B) Histopathology of liver tissues (H&E staining; original magnification, ×400, n = 5/group) at 72 h post-MHV-3 infection was evaluated in the two groups of MHV-3-infected BALB/cJ mice. Livers were collected from saline-treated (a) and CC10-treated (b) BALB/cJ mice at 72 h after MHV-3 infection. Arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation.", "Arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation. (C) Effect of CC10 on serum ALT and AST levels (n = 6-8/group). Values represent means and standard error of three independent experiments performed in triplicate. **P < 0.01 compared with the saline group.", "**P < 0.01 compared with the saline group. six-well plates with DMEM supplemented with 10% FBS until 70-80% confluence. 50 pmol HBP1-siRNA was mixed with 125 µl serum-free DMEM. Two microliter Lipofectamine 2,000 was gently mixed with serum-free DMEM.", "Two microliter Lipofectamine 2,000 was gently mixed with serum-free DMEM. After incubation at 27 • C for 5 min, the solution was added to HUVECs and incubated at 37 • C. Four hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, cells were collected for real-time PCR and western blot analysis to evaluate the effects of HBP1 on Fgl2.", "At 48 h after transfection, cells were collected for real-time PCR and western blot analysis to evaluate the effects of HBP1 on Fgl2. At 24 h after transfection, the CC10 group was supplemented with the CC10 protein (150 ng/mL). After 4 h of stimulation, IFN-γ (10 ng/mL) was added to these cells.", "After 4 h of stimulation, IFN-γ (10 ng/mL) was added to these cells. These cells were then cultured for 24 h before they were harvested for real-time PCR studies to evaluate the effects of CC10 on Fgl2 by HBP1. Negative control was used as a control.", "Negative control was used as a control. To detect whether there was a potential interaction between CC10 protein and Fgl2, CHO cells were transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2 for 48 h. Cells transfected with empty plasmid pcDNA3.1 (mock) were used as negative controls for CC10 gene transfection. Immunoprecipitation and immunoblotting were performed by using Pierce Co-Immunoprecipitation Kit (Pierce, USA).", "Immunoprecipitation and immunoblotting were performed by using Pierce Co-Immunoprecipitation Kit (Pierce, USA). Total cell proteins were extracted as previously described (25) . The proteins were immunoprecipitated by mouse anti-human Fgl2 antibody (1:500, Abnova).", "The proteins were immunoprecipitated by mouse anti-human Fgl2 antibody (1:500, Abnova). For co-immunoprecipitation experiments, western blotting was performed using both rat anti-human uteroglobin/SCGB1A1 Antibody (1:750, R&D, USA) Frontiers in Immunology | and mouse anti-human Fgl2 antibody (1:500, Abnova). Control isotype rat IgG1 was used as a negative control for primary antibodies.", "Control isotype rat IgG1 was used as a negative control for primary antibodies. The human CC10 coding region gene, including a 389 bp sequence, was amplified from homogenized human turbinate tissue by RT-PCR. In this study, the sequences of PCR primers for CC10 were as follows: hCC10-forward, 5 ′ -CCC TCC ACC ATG AAA CTCG-3 ′ ; hCC10-reverse, 5 ′ -TGA GAT GCT TGT GGT TTA TTG AAG-3 ′ .", "In this study, the sequences of PCR primers for CC10 were as follows: hCC10-forward, 5 ′ -CCC TCC ACC ATG AAA CTCG-3 ′ ; hCC10-reverse, 5 ′ -TGA GAT GCT TGT GGT TTA TTG AAG-3 ′ . The PCR products were cloned into pEASY-T1 cloning vector (TransGEN, Beijing, China) and then subcloned into HindIII/XbaI site of pcDNA3.1 vector (Invitrogen, USA) to form eukaryotic expression plasmids pcDNA3.1-hCC10. Microarray analysis was used to screen changes in genome-wide gene expression patterns in THP-1 cells with or without CC10 protein.", "Microarray analysis was used to screen changes in genome-wide gene expression patterns in THP-1 cells with or without CC10 protein. The changes in over 47,000 human gene expression patterns were assessed using Affymetrix gene microarrays (Human Genome U133 Plus 2.0) (CapitalBio Co.,Ltd., Beijing, China). Three replicates were used for microarrays analysis.", "Three replicates were used for microarrays analysis. Data obtained from the experiments are expressed as means ± SEM. Survival curve comparisons were performed with the Log Rank test. Multiple group analyses for data were evaluated by one-way analyses of variance. Analyses of two group results were performed using Student's t-test to evaluate the statistical significance of differences.", "Analyses of two group results were performed using Student's t-test to evaluate the statistical significance of differences. Values of P < 0.05 indicated significance. To establish an animal model of mouse FH, MHV-3 was injected intraperitoneally to BALB/cJ mice (24 mice/group).", "To establish an animal model of mouse FH, MHV-3 was injected intraperitoneally to BALB/cJ mice (24 mice/group). To further study the role of CC10 in FH, recombinant mouse CC10 protein (2 µg/mouse) or saline was administrated into the tail vein 24 h prior to MHV-3 infection. The same dose of CC10 protein or saline was then administered 24 h later.", "The same dose of CC10 protein or saline was then administered 24 h later. The survival rate of the CC10 and saline groups was observed for 10 days. The results showed that mice in the two groups began to die at 48 h after injection of MHV-3 and exhibited symptoms of horripilation, slow activity, and reduced food consumption.", "The results showed that mice in the two groups began to die at 48 h after injection of MHV-3 and exhibited symptoms of horripilation, slow activity, and reduced food consumption. In the CC10 group 24 mice were alive on day 3 after infection, 4 mice alive on day 4, and 3 of 24 (12.5%) mice recovered from fulminant viral hepatitis. At the same time, in saline treated group, there were 5 mice alive on day 3, 1 mice alive on day 4 after infection, and no mice survived to day 5.", "At the same time, in saline treated group, there were 5 mice alive on day 3, 1 mice alive on day 4 after infection, and no mice survived to day 5. That is to say, the mice in the saline group died within 3 or 4 days. Three of 24 (12.5%) mice of the CC10 group recovered from fulminant viral hepatitis ( Figure 1A) .", "Three of 24 (12.5%) mice of the CC10 group recovered from fulminant viral hepatitis ( Figure 1A) . To better understand the mechanisms underlying the biological effects of the CC10 protein, liver function (ALT and AST levels in serum) and liver histology in mice of MHV-3-infected was performed. Liver tissues were harvested 72 h following MHV-3 infection, and liver histology was detected by H&E staining.", "Liver tissues were harvested 72 h following MHV-3 infection, and liver histology was detected by H&E staining. These results showed that there was substantial inflammatory cell infiltration and widespread necrosis of hepatocytes in the liver tissue of the saline group mice (Figure 1Ba ). There were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the CC10 group 72 h after MHV-3 infection (Figure 1Bb) .", "There were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the CC10 group 72 h after MHV-3 infection (Figure 1Bb) . Serum ALT and AST levels in mice were observed 72 h after MHV-3 infection. The results showed that serum ALT and AST levels in the saline group reached a peak 72 h after MHV-3 infection, but there was no significant increase in the CC10 group compared to the levels in the control group (P < 0.01, Figure 1C) .", "The results showed that serum ALT and AST levels in the saline group reached a peak 72 h after MHV-3 infection, but there was no significant increase in the CC10 group compared to the levels in the control group (P < 0.01, Figure 1C) . These results suggested that CC10 protein has a role in protection against MHV-3-induced liver injury in mice. To further elucidate the mechanisms of reduced liver injury following CC10 protein injection, we investigated the cytokines TNF-α and IL-1β expression.", "To further elucidate the mechanisms of reduced liver injury following CC10 protein injection, we investigated the cytokines TNF-α and IL-1β expression. Because these two cytokines play a crucial role in the liver damage of FH. They are characterized by an increase in apoptosis.", "They are characterized by an increase in apoptosis. Levels of TNF-α and IL-1β in liver tissues were markedly reduced in the CC10 group (as shown in Figure 2A) . Hepatic apoptosis (Figure 2B ) was significantly reduced in the CC10 group.", "Hepatic apoptosis (Figure 2B ) was significantly reduced in the CC10 group. We and collaborators have a long standing interest in studying the role of fgl2 in viral hepatitis. Fgl2 has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports.", "Fgl2 has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports. We have provided liver pathology figures and liver function for MHV-3 infected mice with a fgl2 gene knockout as shown in Supplementary Figure 1 . The data was comparable with previous reports from our center and collaborators.", "The data was comparable with previous reports from our center and collaborators. From this current study we shown that CC10 plays a protective role in liver damage.To study the related molecules of CC10 in MHV-3-induced FH mice, we evaluated whether there was crosstalk between Fgl2 and CC10. We found that the expression of Fgl2 in the liver of mice was reduced 72 h after MHV-3 infection and treatment with CC10 protein (Figures 3A,B) .", "We found that the expression of Fgl2 in the liver of mice was reduced 72 h after MHV-3 infection and treatment with CC10 protein (Figures 3A,B) . Furthermore, fibrin deposition, an indicator of liver injury associated with Fgl2 expression in FH, was also decreased in the livers of CC10-treated mice compared to that in controls (Figure 3C ). This indicates that CC10 treatment reduced liver injury after viral infection by inhibiting Fgl2 expression.", "This indicates that CC10 treatment reduced liver injury after viral infection by inhibiting Fgl2 expression. We examined the effect of increasing doses of CC10 protein (0, 50, 150, and 300 ng/mL) on IFN-γ-induced Fgl2 expression in THP-1 cells. CC10 treatment showed a 10.1% decrease in THP-1 cells compared to that in control after stimulation with 10 ng/mL IFN-γ for 12 h. CC10 protein inhibited Fgl2 expression between doses of 0 ng/mL and 300 ng/mL (Figure 4A ).", "CC10 treatment showed a 10.1% decrease in THP-1 cells compared to that in control after stimulation with 10 ng/mL IFN-γ for 12 h. CC10 protein inhibited Fgl2 expression between doses of 0 ng/mL and 300 ng/mL (Figure 4A ). In particular, 150 ng/mL CC10 protein had the strongest inhibitory effect on Fgl2 expression among the doses, and we chose this dose for the following experiments. We explored the effect of different time points of stimulation with a concentration of 150 ng/mL CC10 protein.", "We explored the effect of different time points of stimulation with a concentration of 150 ng/mL CC10 protein. After stimulation with CC10 protein for 6, 12, and 24 h compared to the PBS control, the strongest inhibitory effect on Fgl2 expression was noted at 12 h; hence, we chose this time point for the following studies ( Figure 4B ). An increasing number of studies suggest that macrophages are the primary source of Fgl2.", "An increasing number of studies suggest that macrophages are the primary source of Fgl2. In order to ascertain that CC10 has a direct effect on macrophages, we treated THP-1 cells with recombinant CC10 and assessed the expression of Fgl2. Unlike in controls, IFN-γ induced a significant increase in Fgl2 expression.", "Unlike in controls, IFN-γ induced a significant increase in Fgl2 expression. This effect was attenuated when cells were treated with CC10 protein (Figures 4C,D) , revealing that CC10 directly reduces the levels of Fgl2 in macrophages. To further explore the possibility that CC10 protein directly acts on macrophages, we infected murine PEMs with MHV-3 in the presence of recombinant CC10 and determined Fgl2 expression.", "To further explore the possibility that CC10 protein directly acts on macrophages, we infected murine PEMs with MHV-3 in the presence of recombinant CC10 and determined Fgl2 expression. Compared to levels in the controls, MHV-3infected macrophages exhibited a significant increase in Fgl2 production, and this effect was abolished by using CC10 protein (Figures 5A,B) , indicating that CC10 directly modulates Fgl2 production in macrophages. In order to determine genes that were downregulated after stimulation by CC10 protein, we used DNA microarray analysis to screen for differentially expressed genes.", "In order to determine genes that were downregulated after stimulation by CC10 protein, we used DNA microarray analysis to screen for differentially expressed genes. THP-1 cells were cultured and PMA was added to induce differentiation into macrophages. The production of Fgl2 was stimulated by IFNγ.", "The production of Fgl2 was stimulated by IFNγ. The experimental group was treated with CC10 protein for microarray detection of differentially expressed genes. The results showed that the most obviously downregulated genes were UBE2W, HECTD1, MIR612, ATRX, SOX4, HBP1, and Fgl2 (Supplementary Table 1) .", "The results showed that the most obviously downregulated genes were UBE2W, HECTD1, MIR612, ATRX, SOX4, HBP1, and Fgl2 (Supplementary Table 1) . And then these genes were tested by qPCR. However, UBE2W, HECTD1, MIR612, ATRX, and SOX4 was not differentially expressed by qPCR, while HBP1 and fgl2 were still down-regulated genes.", "However, UBE2W, HECTD1, MIR612, ATRX, and SOX4 was not differentially expressed by qPCR, while HBP1 and fgl2 were still down-regulated genes. DNA microarray analysis identified HBP1 as a down-regulated gene involved in the pathological processes of the regulation of CC10. Recently, very limited studies have explored the role of HBP1 in FH.", "Recently, very limited studies have explored the role of HBP1 in FH. Nevertheless, the mechanistic functions of HBP1 in FH remain largely unexplored. Therefore, we selected this gene for further study.", "Therefore, we selected this gene for further study. qPCR analysis confirmed that mRNA levels of HBP1 were significantly decreased in THP-1 cells after CC10\n\nprotein stimulation compared to that in the PBS control group (Figure 6A ). We knocked down HBP1 using HBP1-siRNA. Then, transfection of HBP1-SiRNA into HUVECs was detected by qPCR and western-blotting methods.", "Then, transfection of HBP1-SiRNA into HUVECs was detected by qPCR and western-blotting methods. As expected, HBP1 knockdown led to significantly decreased expression of HBP1 (Figures 6B,C) . Furthermore, HBP1 knockdown impaired expression of Fgl2 (Figure 6D ), suggesting that HBP1 was able to activate Fgl2. HBP1-SiRNA was used to transfect HUVECs.", "HBP1-SiRNA was used to transfect HUVECs. Then, IFN-γ was added to induce the expression of Fgl2 followed by stimulation with CC10 protein (150 ng/ml) after 2 h. Finally, we explored the expression of Fgl2 by qPCR. The results showed that HBP1-SiRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in HUVECs (Figure 7) .", "The results showed that HBP1-SiRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in HUVECs (Figure 7) . That is to say, CC10 could suppress Fgl2 expression in macrophages. Such an effect may be mediated by the transcription factor HBP1. It is well-known that CC10 protein can suppress the immune response.", "It is well-known that CC10 protein can suppress the immune response. In animal models of allergic diseases of the respiratory tract, most of evidences confirm this inhibition (26) . Its function in FH has not been investigated yet.", "Its function in FH has not been investigated yet. Here, we used a murine FH model established by MHV-3 infection to explore the effects of CC10 in this disease process. To determine the role of CC10 in the pathogenesis of FH, CC10 protein was injected into a mouse FH model established by MHV-3 infection.", "To determine the role of CC10 in the pathogenesis of FH, CC10 protein was injected into a mouse FH model established by MHV-3 infection. MHV-3-induced liver injury in CC10-treated mice occurred rarely and the areas of lesions were much fewer than those in saline-treated control mice. In summary, these results suggested that CC10 could reduce pathological liver damage in this FH model together with lower mortality rates followed by MHV-3 infection.", "In summary, these results suggested that CC10 could reduce pathological liver damage in this FH model together with lower mortality rates followed by MHV-3 infection. MHV-3 induced fulminant viral hepatitis progresses rapidly and infected mice die within 3-5 days. Previous studies suggested fgl2 played a vital role in this process with a 15-40% increase of survival when fgl2 was deleted (12, 15, 27, 28) .", "Previous studies suggested fgl2 played a vital role in this process with a 15-40% increase of survival when fgl2 was deleted (12, 15, 27, 28) . Multiple inflammatory factors or mediators including TNF-α and IFN-γ, IL-1β and C5aR have been demonstrated to promote FH progression with significant discrepancies between liver damage and survival rate (29) (30) (31) (32) , which is accordant with our observation that CC10 substantially alleviated liver injury though survival rate improved mildly. The survival rate based on hours may be more accurate to examine the effect of CC10 on FH.", "The survival rate based on hours may be more accurate to examine the effect of CC10 on FH. It is speculated that fgl2 can mediate lethality in MHV-3-induced FH. This is due to the fact that fgl2 induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity (15) .", "This is due to the fact that fgl2 induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity (15) . To determine whether the tissue necrosis was mediated by Fgl2 in CC10-treated mice following infection, Fgl2 expression was observed. Results suggested that the expression of Fgl2 was significantly increased in MHV-3-induced FH mice and CC10 treatment significantly reduced the production of Fgl2 in the infected liver and serum.", "Results suggested that the expression of Fgl2 was significantly increased in MHV-3-induced FH mice and CC10 treatment significantly reduced the production of Fgl2 in the infected liver and serum. In addition, decreased fibrinogen deposition was also observed in the livers of CC10-treated mice. Therefore, our research results strongly clarify that the lower mortality of CC10-treated mice after MHV-3 infection is due to the lower levels of Fgl2 and decreased fibrinogen deposition.", "Therefore, our research results strongly clarify that the lower mortality of CC10-treated mice after MHV-3 infection is due to the lower levels of Fgl2 and decreased fibrinogen deposition. Indeed, it has been reported that Fgl2 is expressed on macrophages, and the expression of Fgl2 is believed to be induced by IFN-γ and TNF-α (22) . Cultured THP-1 cells activated by IFN-γ or IL-2 have been demonstrated, with induction of Fgl2 expression and enhanced activation of human prothrombin (23) .", "Cultured THP-1 cells activated by IFN-γ or IL-2 have been demonstrated, with induction of Fgl2 expression and enhanced activation of human prothrombin (23) . Therefore, in this study, we explored this cell line to investigate the modulation of CC10 on Fgl2. Surprisingly, we found that CC10 directly inhibited IFN-γ-induced Fgl2 expression in THP-1 cells.", "Surprisingly, we found that CC10 directly inhibited IFN-γ-induced Fgl2 expression in THP-1 cells. As we know, IFN-γ has proved to be the main cytokine that leads to the development and progression of FH. Also, it was shown that IFN-γ might exert its own proinflammatory biological function through enhancing Fgl2 expression.", "Also, it was shown that IFN-γ might exert its own proinflammatory biological function through enhancing Fgl2 expression. Therefore, in our study, CC10 might counter the effect of IFN-γ in the setting of FH, which substantiates its role in FH. These results demonstrated that CC10 regulates the expression of Fgl2 in macrophages.", "These results demonstrated that CC10 regulates the expression of Fgl2 in macrophages. In the current study, we used co-immunoprecipitation to analyze binding between CC10 and Fgl2. In this study, we investigated possible protein-protein interactions between CC10 and Fgl2 in vitro. The Chinese hamster ovary (CHO) cells transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2.", "The Chinese hamster ovary (CHO) cells transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2. Cellular proteins were immunoprecipitated with anti-CC10 antibody or anti-Fgl2 antibody. Immunoblotting was performed with anti-Fgl2 and anti-CC10 antibodies.", "Immunoblotting was performed with anti-Fgl2 and anti-CC10 antibodies. Immunoprecipitation of protein extracts from pcDNA 3.1-CC10 and pcDNA3.1-Fgl2 co-transfected CHO cells with anti-Fgl2 or anti-CC10 antibody followed by western blotting with Fgl2 and CC10 antibodies indicated that CC10 did not co-immunoprecipitate with Fgl2, showing that there is no direct relationship between CC10 and Fgl2 (data not shown). The results showed that CC10 has no direct interaction with Fgl2.", "The results showed that CC10 has no direct interaction with Fgl2. From our previous study the gene of fgl2 contributed profoundly in MHV-3 induced fulminant hepatitis and is extensively expressed in macrophages and endothelium (12, 33) . Our microarray indicated a CC10 down-regulated fgl2 expression and this is further confirmed by qPCR and Western blotting in vivo (peritoneal macrophages) and in vitro (THP-1, macrophage cell line).", "Our microarray indicated a CC10 down-regulated fgl2 expression and this is further confirmed by qPCR and Western blotting in vivo (peritoneal macrophages) and in vitro (THP-1, macrophage cell line). Therefore, it is reasonable to focus on macrophages to display the effect of CC10 on fgl2 expression and eventually mice survival. We entirely agree there may be other possibilities for a protective effect of CC10 to contribute to the disease process.", "We entirely agree there may be other possibilities for a protective effect of CC10 to contribute to the disease process. This is worth further studies. The potential receptor of CC10 has not been revealed yet. Our previous study have demonstrated that CC10 have effect of dendritic cells in allergic rhinitis (34) .", "Our previous study have demonstrated that CC10 have effect of dendritic cells in allergic rhinitis (34) . In this research, we evaluated the effect of CC10 on macrophages functions and found Fgl2 was substantially down-regulated upon CC10 treatment, therefore, we speculate that potential CC10 receptor may be also expressed on macrophages. The potential target of CC10 on other immune cells cannot be excluded.", "The potential target of CC10 on other immune cells cannot be excluded. DNA microarray analysis is one of the most powerful approaches for the potential identification of unexpected genes involved in pathogenic processes. By using this approach, HMGbox transcription factor 1 (HBP1) was found to be one of the most downregulated genes after CC10 treatment of THP-1 cells.", "By using this approach, HMGbox transcription factor 1 (HBP1) was found to be one of the most downregulated genes after CC10 treatment of THP-1 cells. HBP1 is a well-described transcriptional repressor that modulates expression of genes involved in cell cycle progression. In a recent study, it was found that HBP1 is a direct target of miR-21 and confirmed that HBP1 modulates the inhibitory function of miR-21-ASO in hepatosteatosis and carcinogenesis simultaneously (23) .", "In a recent study, it was found that HBP1 is a direct target of miR-21 and confirmed that HBP1 modulates the inhibitory function of miR-21-ASO in hepatosteatosis and carcinogenesis simultaneously (23) . HBP1 is an endogenous inhibitor of the Wnt signaling pathway in both normal and cancer cells. The tumor suppressor role of HBP1 has been reported in some malignancies, such as oral cancer and glioma (35) .", "The tumor suppressor role of HBP1 has been reported in some malignancies, such as oral cancer and glioma (35) . However, an association between HBP1 and Fgl2 has not been investigated yet. The current study clearly demonstrated that CC10 protects against MHV-3 induced FH via suppression of Fgl2 expression. Such effects might be mediated by HBP1.", "Such effects might be mediated by HBP1. However, the functional status of HBP1 in the CC10 pathway requires further research, and such studies are conducting in our laboratory. In conclusion, we demonstrated that CC10 could limit the immunopathological damage in MHV-3-induced FH mice.", "In conclusion, we demonstrated that CC10 could limit the immunopathological damage in MHV-3-induced FH mice. Our results suggest that enhancing CC10 expression by an immunotherapeutic approach might be an effective treatment for FH. HY performed all the described experiments and wrote the manuscript. YL assisted with some experiments, analyzed experimental results, and edited the manuscript.", "YL assisted with some experiments, analyzed experimental results, and edited the manuscript. HW analyzed experimental results. XW reviewed and edited the manuscript. JH, WY, DX, XL, GS, and QN provided experimental help and design." ]

[ "Background: Fulminant hepatitis (FH) is a serious threat to human life, accompanied by massive and rapid necroinflammation. Kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for FH. Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection.", "Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection. Clara cell 10 kDa (CC10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. However, its mechanisms of action and pathogenic roles in other disease are still unclear.", "However, its mechanisms of action and pathogenic roles in other disease are still unclear. In this study, we aimed to determine the role of CC10 in FH and the regulation of Fgl2 by CC10. Methods: A mouse FH model was established by peritoneal injection of MHV-3.", "Methods: A mouse FH model was established by peritoneal injection of MHV-3. The mice received CC10 protein through tail vein injection before viral infection. Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined.", "Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. The regulatory effect of CC10 on Fgl2 expression was investigated using THP-1 cells and mouse peritoneal macrophages in vitro. Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group.", "Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group. Meanwhile, the levels of ALT and AST in serum were significantly decreased and liver damage was reduced. Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein.", "Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein. In vitro, CC10 was found to significantly inhibit the expression of Fgl2 in IFN-γ-treated THP-1 cells and MHV-3-infected mouse peritoneal macrophages by western blot and real-time PCR. However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation.", "However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation. Microarray investigations suggested that HMG-box transcription factor 1 (HBP1) was significantly low in CC10-treated and IFN-γ-primed THP-1 cells. HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs).", "HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs). Conclusion:CC10 protects against MHV-3-induced FH via suppression of Fgl2 expression in macrophages. Such effects may be mediated by the transcription factor HBP1.", "Such effects may be mediated by the transcription factor HBP1. Text: Fulminant hepatitis (FH) is a serious life-threatening disease characterized by massive hepatocyte necrosis, severe liver damage, and high mortality. The underlying mechanisms and the pathogenesis of FH are not clear.", "The underlying mechanisms and the pathogenesis of FH are not clear. However, accumulating evidence suggests that, regardless of the pathogenesis of FH, the host's inflammatory responses contribute to liver microcirculatory disorders and injuries. Accordingly, It has been shown that immune cell activation and inflammatory cytokines play an important role in FH (1) .", "Accordingly, It has been shown that immune cell activation and inflammatory cytokines play an important role in FH (1) . In recent years, our laboratory has conducted extensive research on the pathogenesis of FH and found that immune cells play a key role in it. Kupffer cells, natural killer (NK) cells (2, 3) , cytotoxic T-lymphocytes (CTLs), and double negative T-cells (DNT) (4) (5) (6) in liver and the cytokines that are produced by these cells cause liver damage.", "Kupffer cells, natural killer (NK) cells (2, 3) , cytotoxic T-lymphocytes (CTLs), and double negative T-cells (DNT) (4) (5) (6) in liver and the cytokines that are produced by these cells cause liver damage. Prothrombinase Fgl2 belongs to the fibrinogen superfamily and is produced by activated macrophages or endothelial cells, transforming prothrombin directly into thrombin, so as to quickly initiate the process of coagulation. This promotes the conversion of fibrinogen into fibrin, resulting in thrombosis (7) (8) (9) (10) (11) (12) .", "This promotes the conversion of fibrinogen into fibrin, resulting in thrombosis (7) (8) (9) (10) (11) (12) . Our study found that Fgl2 was highly expressed in peripheral blood mononuclear cells (PBMCs) and in liver tissue of humans or mice with severe viral hepatitis, and was positively related to the severity of the disease (13, 14) . Gene therapy targeting Fgl2 silencing showed that the survival rate of fulminant hepatitis mice increased from 0 to 33.3% (15) .", "Gene therapy targeting Fgl2 silencing showed that the survival rate of fulminant hepatitis mice increased from 0 to 33.3% (15) . Thus far, the discovery and related research involving Fgl2 have provided new insights into the molecular mechanism of hepatocyte necrosis in FH. In view of the important role of Fgl2 in severe viral hepatitis, investigations concerning the regulation of Fgl2 will be beneficial in the search for new strategies for treatment of severe hepatitis.", "In view of the important role of Fgl2 in severe viral hepatitis, investigations concerning the regulation of Fgl2 will be beneficial in the search for new strategies for treatment of severe hepatitis. Clara cell 10 kDa protein (CC10), also considered to be uteroglobin, Clara cell secretory protein, is one of members of secretoglobin superfamily. Expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world (16) .", "Expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world (16) . CC10 has immunomodulatory and anti-inflammatory effects. Compared to wild-type mice, CC10-knockout mice exhibited excessive airway inflammation Abbreviations: FH, fulminant hepatitis; MHV-3, murine hepatitis virus strain 3; Fgl2, Fibrinogen-like protein 2; CC10, Clara cell 10 KDa protein; ALF, acute liver failure; PFU, plaque-forming units; PBS, phosphate-buffered saline; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCA, pro-coagulant activity; HRP, horseradish peroxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.", "Compared to wild-type mice, CC10-knockout mice exhibited excessive airway inflammation Abbreviations: FH, fulminant hepatitis; MHV-3, murine hepatitis virus strain 3; Fgl2, Fibrinogen-like protein 2; CC10, Clara cell 10 KDa protein; ALF, acute liver failure; PFU, plaque-forming units; PBS, phosphate-buffered saline; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCA, pro-coagulant activity; HRP, horseradish peroxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. caused by allergic reaction and bacterial and viral infections (17) . Reduced levels of CC10 are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis (18) (19) (20) (21) .", "Reduced levels of CC10 are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis (18) (19) (20) (21) . Previous studies and published articles show that CC10 protein can not only inhibit Th17 cell responses by inhibiting expression of related molecules of dendritic cells and cytokines in mice with allergic rhinitis, but also can inhibit chitosan-3 like protein 1 (22, 23) . Moreover, CC10 inhibits the expression of an important immune regulator, osteopontin (OPN), in models of allergic rhinitis (21) .", "Moreover, CC10 inhibits the expression of an important immune regulator, osteopontin (OPN), in models of allergic rhinitis (21) . In this study, we investigated the role of CC10 in hepatitis virus strain 3 (MHV-3)-induced FH in mice and explored whether CC10 protein could regulate Fgl2 in the disease process. Female BALB/cJ mice (Shanghai Shilaike Animal Seed Center, Shanghai, China), 6-8 weeks of age, with a body weight of 18.0-20.0 g, were kept in Tongji Hospital with food and water.", "Female BALB/cJ mice (Shanghai Shilaike Animal Seed Center, Shanghai, China), 6-8 weeks of age, with a body weight of 18.0-20.0 g, were kept in Tongji Hospital with food and water. Mice were divided into two groups: CC10 group (experimental group) and phosphate-buffered saline (PBS) group (control group). This study was carried out in accordance with the recommendations of the guidelines of the National Institutes of Health and the Animal Experiment Committee of Tongji hospital.", "This study was carried out in accordance with the recommendations of the guidelines of the National Institutes of Health and the Animal Experiment Committee of Tongji hospital. This study was reviewed and approved by the Animal Experiment Committee of Tongji hospital. The human monocyte cell line THP-1 was purchased from the Cell Institute of the Chinese Academy of Sciences (Shanghai, China).", "The human monocyte cell line THP-1 was purchased from the Cell Institute of the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from the Biology Treasure Center of Wuhan University, China. The Chinese hamster ovary (CHO) cell line was acquired from the typical culture preservation commission cell bank, the Chinese Academy of Sciences (Shanghai, China).", "The Chinese hamster ovary (CHO) cell line was acquired from the typical culture preservation commission cell bank, the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) and CHO cells were cultured in Dulbecco's modified Eagle's medium (DMEM), and THP-1 cells were maintained in RPMI 1,640 containing 10% heat inactivated fetal bovine serum (FBS, Gibco Life Technologies, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin and cultured at 37 • C, 50 mL/L CO 2 and 95% humidity. Peritoneal exudative macrophages (PEMs) were obtained from BALB/cJ mice.", "Peritoneal exudative macrophages (PEMs) were obtained from BALB/cJ mice. Cells were resuspended in RPMI 1,640 supplemented with 10% FBS at 1-2 × 10 6 cells/mL in a 6-well plate and incubated for 4 h. They were then washed with RPMI 1640 medium and non-adherent cells discarded. The adherent cells were macrophages and were incubated for a further 12 h. Peritoneal exudative macrophages (PEMs) were divided into two groups.", "The adherent cells were macrophages and were incubated for a further 12 h. Peritoneal exudative macrophages (PEMs) were divided into two groups. One group was supplemented with CC10 protein (150 ng/mL) and in the other group, PBS was added. After 2 h of stimulation, 1,000 plaque forming units (PFUs) of MHV-3 was added to the cells, which were then cultured for 4 h. Peritoneal exudative macrophages (PEMs) were harvested and lysed for real-time PCR and western blotting analysis.", "After 2 h of stimulation, 1,000 plaque forming units (PFUs) of MHV-3 was added to the cells, which were then cultured for 4 h. Peritoneal exudative macrophages (PEMs) were harvested and lysed for real-time PCR and western blotting analysis. Cell apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method with a TUNEL apoptosis detection kit (Roche, Switzerland). Briefly, 5 µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase K for 30 min at 37 • C. After stopping the proteinase K digestion reaction with PBS, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dUTP at a ratio of 2:29, respectively), for 2 h at 37 • C in an immunohistochemistry wet box.", "Briefly, 5 µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase K for 30 min at 37 • C. After stopping the proteinase K digestion reaction with PBS, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dUTP at a ratio of 2:29, respectively), for 2 h at 37 • C in an immunohistochemistry wet box. Following washing and blocking, each section was supplemented with reagent (converter-POD) to cover the tissues and incubated for 30 min at 37 • C in a wet box. Then, the liver tissue sections were washed with PBS, and colored with diaminobenzidine (DAB) subsequently.", "Then, the liver tissue sections were washed with PBS, and colored with diaminobenzidine (DAB) subsequently. Hepatocytes with nucleus stained brownish yellow were considered to be apoptotic cells. The expression of Fgl2 on THP-1 cells was measured by flow cytometry (BD FACS Canto II, USA).", "The expression of Fgl2 on THP-1 cells was measured by flow cytometry (BD FACS Canto II, USA). Briefly, cells (2 × 10 5 per tube) were incubated with Human TruStrain FcX (Fc Receptor Blocking solution, BioLegend, USA) for 10 min at room temperature and then incubated in the dark with mouse anti-Fgl2 antibody (1:100, Abnova,) or normal goat serum (an isotype control) at 4 • C for 40 min. Cells were washed with PBS and incubated in the dark with PE-conjugated goat anti-mouse IgG antibody (1:50, BioLegend, USA) at 4 • C for 30 min.", "Cells were washed with PBS and incubated in the dark with PE-conjugated goat anti-mouse IgG antibody (1:50, BioLegend, USA) at 4 • C for 30 min. Cells were then washed with PBS and resuspended in 300 µL PBS for study. Liver slices were fixed in 4% paraformaldehyde and then embedded in paraffin.", "Liver slices were fixed in 4% paraformaldehyde and then embedded in paraffin. Immunohistochemistry of liver tissues was performed using SP-9001 SPlink Detection Kits (Biotin-Streptavidin HRP Detection Systems) (ZSGB-BIO, Beijing, China) according to the manufacturer's instructions. For immunohistochemistry staining, the expression of Fgl2, fibrinogen, Fas and TNF-receptor 1 in mouse liver tissues was detected with polyclonal rabbit anti-mouse Fgl2 antibody (1:100, Proteintech, USA), polyclonal rabbit anti-mouse fibrinogen antibody (1:1,000, Abcam, EngLand), polyclonal rabbit antimouse Fas antibody (1:50, Abcam, EngLand), and polyclonal rabbit anti-mouse TNF-receptor 1 antibody (1:500, Abcam, EngLand), respectively.", "For immunohistochemistry staining, the expression of Fgl2, fibrinogen, Fas and TNF-receptor 1 in mouse liver tissues was detected with polyclonal rabbit anti-mouse Fgl2 antibody (1:100, Proteintech, USA), polyclonal rabbit anti-mouse fibrinogen antibody (1:1,000, Abcam, EngLand), polyclonal rabbit antimouse Fas antibody (1:50, Abcam, EngLand), and polyclonal rabbit anti-mouse TNF-receptor 1 antibody (1:500, Abcam, EngLand), respectively. After incubation with an horseradish peroxidase (HRP)-labeled goat IgG fraction to rabbit IgG Fc, the target protein was detected using a DAB kit (ZSGB-BIO, Beijing, China). The slides were then counterstained with hematoxylin and visualized under a microscope (Olympus, Tokyo, Japan).", "The slides were then counterstained with hematoxylin and visualized under a microscope (Olympus, Tokyo, Japan). Liver tissue and cells were homogenized in RIPA lysis buffer with phenyl methane sulfonyl fluoride (PMSF) protease inhibitor. Protein lysates were separated by SDS-PAGE, and western blotting was performed using a monoclonal mouse antihuman/mouse Fgl2 (1:750, Abnova), a monoclonal mouse antihuman HBP1 (1:100, Santa Cruz, USA), and a monoclonal rabbit anti-human/mouse β-actin (1:1,000, Cell Signaling Technology, USA).", "Protein lysates were separated by SDS-PAGE, and western blotting was performed using a monoclonal mouse antihuman/mouse Fgl2 (1:750, Abnova), a monoclonal mouse antihuman HBP1 (1:100, Santa Cruz, USA), and a monoclonal rabbit anti-human/mouse β-actin (1:1,000, Cell Signaling Technology, USA). Liver tissues were collected from MHV-3-infected BALB/cJ mice at 72 h, and total RNA was extracted using Trizol Reagent (Invitrogen, USA) and then reverse transcribed into cDNA by using ReverTra Ace qPCR RT kit (TOYOBO, Japan). The cDNA was then amplified by RT-PCR by using Dream Taq Green PCR Master Mix (2 ×) (Thermo Scientific, USA).", "The cDNA was then amplified by RT-PCR by using Dream Taq Green PCR Master Mix (2 ×) (Thermo Scientific, USA). Realtime quantitative PCR (qPCR) with SYBR Green Real-time PCR Master Mix (TOYOBO, Japan) was performed using a CFX96 real-time PCR detection system (Bio-Rad, USA) and mRNA levels were normalized with reference to those of the house keeping gene GAPDH. Primer sequences for qPCR amplification were as follows: mTNF-α forward, 5 ′ -TTT GAG ATC CAT GCC GTT GG-3 ′ ; mTNF-α reverse, 5 ′ -GCCA CCA CGC TCT TCT GT-3 ′ ; mIL-1β forward, 5 ′ -TGT AAT GAA AGA CGG CAC ACC-3 ′ ; mIL-1β reverse, 5 ′ -TCT TCT TTG GGT ATT GCT TGG-3 ′ .", "Primer sequences for qPCR amplification were as follows: mTNF-α forward, 5 ′ -TTT GAG ATC CAT GCC GTT GG-3 ′ ; mTNF-α reverse, 5 ′ -GCCA CCA CGC TCT TCT GT-3 ′ ; mIL-1β forward, 5 ′ -TGT AAT GAA AGA CGG CAC ACC-3 ′ ; mIL-1β reverse, 5 ′ -TCT TCT TTG GGT ATT GCT TGG-3 ′ . mFgl2 forward, 5 ′ -GCC AAA TGT GAG TCC CTG GAA-3 ′ ; mFgl2 reverse, 5 ′ -TTC CAC CCA AGA GCA CGT TTA AG-3 ′ ; hFgl2 forward 5 ′ -ACA GTT CAG GCT GGT GGT-3 ′ ; hFgl2 reverse, 5 ′ -GGC TTA AAG TGC TTG GGT-3 ′ ; HBP1 forward, 5 ′ -TGA AGC AGA AGC TGG GAGT-3 ′ ; HBP1 reverse,\n\nTHP-1 cells were treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma, USA) for 48 h to induce differentiation toward adherent macrophage-like cells as reported previously (24) . The CC10 group was supplemented with CC10 protein (150 ng/ml).", "The CC10 group was supplemented with CC10 protein (150 ng/ml). After 2 h of stimulation, IFN-γ (10 ng/ml) was added to these cells, which were then cultured for 12 h before they were collected for western blotting and real-time PCR studies. The Chinese hamster ovary (CHO) cells were cultured in 10 cm cell culture dishes with DMEM supplemented with 10% FBS until 80-90% confluence.", "The Chinese hamster ovary (CHO) cells were cultured in 10 cm cell culture dishes with DMEM supplemented with 10% FBS until 80-90% confluence. Next, 12 µg pcDNA3.1-hFgl2 (constructed in our lab) was mixed with 12 µg pcDNA3.1-hCC10 in serumfree DMEM. The mixture was then combined with Lipofectamine 2,000 (Invitrogen, USA) and mixed gently.", "The mixture was then combined with Lipofectamine 2,000 (Invitrogen, USA) and mixed gently. After incubation at 27 • C for 20 min, the solution was added to CHO cells and incubated at 37 • C in 5% CO 2 . Four to Six hour after transfection, the medium was removed and fresh medium containing 10% FBS was added.", "Four to Six hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, the cells were collected for co-immunoprecipitation analysis to evaluate the interaction of CC10 with Fgl2. Both HUVEC and THP-1 cells express fgl2.", "Both HUVEC and THP-1 cells express fgl2. However, in the transfection experiments, it is difficult to transfect the THP-1 cells with siRNA, so we use HUVEC instead of THP-1. Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in FIGURE 1 | CC10 protein increased survival rate and reduced liver damage in mice.", "Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in FIGURE 1 | CC10 protein increased survival rate and reduced liver damage in mice. (A) The survival rate of CC10 group is higher than the control group comprised of MHV-3-infected BALB/cJ mice treated with saline. CC10 protein (2 µg) or saline were injected into mice by tail vein.", "CC10 protein (2 µg) or saline were injected into mice by tail vein. BALB/cJ mice then received 100 PFU of MHV-3 intraperitoneally 24 h later to develop fulminant viral hepatitis. Then, CC10 protein (2 µg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later.", "Then, CC10 protein (2 µg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later. The survival rate was observed for 10 days (n = 24/group). Representative data from three independent experiments are shown. The survival curve was analyzed by using the Log-Rank Test.", "The survival curve was analyzed by using the Log-Rank Test. ***P < 0.001 compared with saline group. (B) Histopathology of liver tissues (H&E staining; original magnification, ×400, n = 5/group) at 72 h post-MHV-3 infection was evaluated in the two groups of MHV-3-infected BALB/cJ mice.", "(B) Histopathology of liver tissues (H&E staining; original magnification, ×400, n = 5/group) at 72 h post-MHV-3 infection was evaluated in the two groups of MHV-3-infected BALB/cJ mice. Livers were collected from saline-treated (a) and CC10-treated (b) BALB/cJ mice at 72 h after MHV-3 infection. Arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation.", "Arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation. (C) Effect of CC10 on serum ALT and AST levels (n = 6-8/group). Values represent means and standard error of three independent experiments performed in triplicate. **P < 0.01 compared with the saline group.", "**P < 0.01 compared with the saline group. six-well plates with DMEM supplemented with 10% FBS until 70-80% confluence. 50 pmol HBP1-siRNA was mixed with 125 µl serum-free DMEM. Two microliter Lipofectamine 2,000 was gently mixed with serum-free DMEM.", "Two microliter Lipofectamine 2,000 was gently mixed with serum-free DMEM. After incubation at 27 • C for 5 min, the solution was added to HUVECs and incubated at 37 • C. Four hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, cells were collected for real-time PCR and western blot analysis to evaluate the effects of HBP1 on Fgl2.", "At 48 h after transfection, cells were collected for real-time PCR and western blot analysis to evaluate the effects of HBP1 on Fgl2. At 24 h after transfection, the CC10 group was supplemented with the CC10 protein (150 ng/mL). After 4 h of stimulation, IFN-γ (10 ng/mL) was added to these cells.", "After 4 h of stimulation, IFN-γ (10 ng/mL) was added to these cells. These cells were then cultured for 24 h before they were harvested for real-time PCR studies to evaluate the effects of CC10 on Fgl2 by HBP1. Negative control was used as a control.", "Negative control was used as a control. To detect whether there was a potential interaction between CC10 protein and Fgl2, CHO cells were transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2 for 48 h. Cells transfected with empty plasmid pcDNA3.1 (mock) were used as negative controls for CC10 gene transfection. Immunoprecipitation and immunoblotting were performed by using Pierce Co-Immunoprecipitation Kit (Pierce, USA).", "Immunoprecipitation and immunoblotting were performed by using Pierce Co-Immunoprecipitation Kit (Pierce, USA). Total cell proteins were extracted as previously described (25) . The proteins were immunoprecipitated by mouse anti-human Fgl2 antibody (1:500, Abnova).", "The proteins were immunoprecipitated by mouse anti-human Fgl2 antibody (1:500, Abnova). For co-immunoprecipitation experiments, western blotting was performed using both rat anti-human uteroglobin/SCGB1A1 Antibody (1:750, R&D, USA) Frontiers in Immunology | and mouse anti-human Fgl2 antibody (1:500, Abnova). Control isotype rat IgG1 was used as a negative control for primary antibodies.", "Control isotype rat IgG1 was used as a negative control for primary antibodies. The human CC10 coding region gene, including a 389 bp sequence, was amplified from homogenized human turbinate tissue by RT-PCR. In this study, the sequences of PCR primers for CC10 were as follows: hCC10-forward, 5 ′ -CCC TCC ACC ATG AAA CTCG-3 ′ ; hCC10-reverse, 5 ′ -TGA GAT GCT TGT GGT TTA TTG AAG-3 ′ .", "In this study, the sequences of PCR primers for CC10 were as follows: hCC10-forward, 5 ′ -CCC TCC ACC ATG AAA CTCG-3 ′ ; hCC10-reverse, 5 ′ -TGA GAT GCT TGT GGT TTA TTG AAG-3 ′ . The PCR products were cloned into pEASY-T1 cloning vector (TransGEN, Beijing, China) and then subcloned into HindIII/XbaI site of pcDNA3.1 vector (Invitrogen, USA) to form eukaryotic expression plasmids pcDNA3.1-hCC10. Microarray analysis was used to screen changes in genome-wide gene expression patterns in THP-1 cells with or without CC10 protein.", "Microarray analysis was used to screen changes in genome-wide gene expression patterns in THP-1 cells with or without CC10 protein. The changes in over 47,000 human gene expression patterns were assessed using Affymetrix gene microarrays (Human Genome U133 Plus 2.0) (CapitalBio Co.,Ltd., Beijing, China). Three replicates were used for microarrays analysis.", "Three replicates were used for microarrays analysis. Data obtained from the experiments are expressed as means ± SEM. Survival curve comparisons were performed with the Log Rank test. Multiple group analyses for data were evaluated by one-way analyses of variance. Analyses of two group results were performed using Student's t-test to evaluate the statistical significance of differences.", "Analyses of two group results were performed using Student's t-test to evaluate the statistical significance of differences. Values of P < 0.05 indicated significance. To establish an animal model of mouse FH, MHV-3 was injected intraperitoneally to BALB/cJ mice (24 mice/group).", "To establish an animal model of mouse FH, MHV-3 was injected intraperitoneally to BALB/cJ mice (24 mice/group). To further study the role of CC10 in FH, recombinant mouse CC10 protein (2 µg/mouse) or saline was administrated into the tail vein 24 h prior to MHV-3 infection. The same dose of CC10 protein or saline was then administered 24 h later.", "The same dose of CC10 protein or saline was then administered 24 h later. The survival rate of the CC10 and saline groups was observed for 10 days. The results showed that mice in the two groups began to die at 48 h after injection of MHV-3 and exhibited symptoms of horripilation, slow activity, and reduced food consumption.", "The results showed that mice in the two groups began to die at 48 h after injection of MHV-3 and exhibited symptoms of horripilation, slow activity, and reduced food consumption. In the CC10 group 24 mice were alive on day 3 after infection, 4 mice alive on day 4, and 3 of 24 (12.5%) mice recovered from fulminant viral hepatitis. At the same time, in saline treated group, there were 5 mice alive on day 3, 1 mice alive on day 4 after infection, and no mice survived to day 5.", "At the same time, in saline treated group, there were 5 mice alive on day 3, 1 mice alive on day 4 after infection, and no mice survived to day 5. That is to say, the mice in the saline group died within 3 or 4 days. Three of 24 (12.5%) mice of the CC10 group recovered from fulminant viral hepatitis ( Figure 1A) .", "Three of 24 (12.5%) mice of the CC10 group recovered from fulminant viral hepatitis ( Figure 1A) . To better understand the mechanisms underlying the biological effects of the CC10 protein, liver function (ALT and AST levels in serum) and liver histology in mice of MHV-3-infected was performed. Liver tissues were harvested 72 h following MHV-3 infection, and liver histology was detected by H&E staining.", "Liver tissues were harvested 72 h following MHV-3 infection, and liver histology was detected by H&E staining. These results showed that there was substantial inflammatory cell infiltration and widespread necrosis of hepatocytes in the liver tissue of the saline group mice (Figure 1Ba ). There were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the CC10 group 72 h after MHV-3 infection (Figure 1Bb) .", "There were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the CC10 group 72 h after MHV-3 infection (Figure 1Bb) . Serum ALT and AST levels in mice were observed 72 h after MHV-3 infection. The results showed that serum ALT and AST levels in the saline group reached a peak 72 h after MHV-3 infection, but there was no significant increase in the CC10 group compared to the levels in the control group (P < 0.01, Figure 1C) .", "The results showed that serum ALT and AST levels in the saline group reached a peak 72 h after MHV-3 infection, but there was no significant increase in the CC10 group compared to the levels in the control group (P < 0.01, Figure 1C) . These results suggested that CC10 protein has a role in protection against MHV-3-induced liver injury in mice. To further elucidate the mechanisms of reduced liver injury following CC10 protein injection, we investigated the cytokines TNF-α and IL-1β expression.", "To further elucidate the mechanisms of reduced liver injury following CC10 protein injection, we investigated the cytokines TNF-α and IL-1β expression. Because these two cytokines play a crucial role in the liver damage of FH. They are characterized by an increase in apoptosis.", "They are characterized by an increase in apoptosis. Levels of TNF-α and IL-1β in liver tissues were markedly reduced in the CC10 group (as shown in Figure 2A) . Hepatic apoptosis (Figure 2B ) was significantly reduced in the CC10 group.", "Hepatic apoptosis (Figure 2B ) was significantly reduced in the CC10 group. We and collaborators have a long standing interest in studying the role of fgl2 in viral hepatitis. Fgl2 has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports.", "Fgl2 has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports. We have provided liver pathology figures and liver function for MHV-3 infected mice with a fgl2 gene knockout as shown in Supplementary Figure 1 . The data was comparable with previous reports from our center and collaborators.", "The data was comparable with previous reports from our center and collaborators. From this current study we shown that CC10 plays a protective role in liver damage.To study the related molecules of CC10 in MHV-3-induced FH mice, we evaluated whether there was crosstalk between Fgl2 and CC10. We found that the expression of Fgl2 in the liver of mice was reduced 72 h after MHV-3 infection and treatment with CC10 protein (Figures 3A,B) .", "We found that the expression of Fgl2 in the liver of mice was reduced 72 h after MHV-3 infection and treatment with CC10 protein (Figures 3A,B) . Furthermore, fibrin deposition, an indicator of liver injury associated with Fgl2 expression in FH, was also decreased in the livers of CC10-treated mice compared to that in controls (Figure 3C ). This indicates that CC10 treatment reduced liver injury after viral infection by inhibiting Fgl2 expression.", "This indicates that CC10 treatment reduced liver injury after viral infection by inhibiting Fgl2 expression. We examined the effect of increasing doses of CC10 protein (0, 50, 150, and 300 ng/mL) on IFN-γ-induced Fgl2 expression in THP-1 cells. CC10 treatment showed a 10.1% decrease in THP-1 cells compared to that in control after stimulation with 10 ng/mL IFN-γ for 12 h. CC10 protein inhibited Fgl2 expression between doses of 0 ng/mL and 300 ng/mL (Figure 4A ).", "CC10 treatment showed a 10.1% decrease in THP-1 cells compared to that in control after stimulation with 10 ng/mL IFN-γ for 12 h. CC10 protein inhibited Fgl2 expression between doses of 0 ng/mL and 300 ng/mL (Figure 4A ). In particular, 150 ng/mL CC10 protein had the strongest inhibitory effect on Fgl2 expression among the doses, and we chose this dose for the following experiments. We explored the effect of different time points of stimulation with a concentration of 150 ng/mL CC10 protein.", "We explored the effect of different time points of stimulation with a concentration of 150 ng/mL CC10 protein. After stimulation with CC10 protein for 6, 12, and 24 h compared to the PBS control, the strongest inhibitory effect on Fgl2 expression was noted at 12 h; hence, we chose this time point for the following studies ( Figure 4B ). An increasing number of studies suggest that macrophages are the primary source of Fgl2.", "An increasing number of studies suggest that macrophages are the primary source of Fgl2. In order to ascertain that CC10 has a direct effect on macrophages, we treated THP-1 cells with recombinant CC10 and assessed the expression of Fgl2. Unlike in controls, IFN-γ induced a significant increase in Fgl2 expression.", "Unlike in controls, IFN-γ induced a significant increase in Fgl2 expression. This effect was attenuated when cells were treated with CC10 protein (Figures 4C,D) , revealing that CC10 directly reduces the levels of Fgl2 in macrophages. To further explore the possibility that CC10 protein directly acts on macrophages, we infected murine PEMs with MHV-3 in the presence of recombinant CC10 and determined Fgl2 expression.", "To further explore the possibility that CC10 protein directly acts on macrophages, we infected murine PEMs with MHV-3 in the presence of recombinant CC10 and determined Fgl2 expression. Compared to levels in the controls, MHV-3infected macrophages exhibited a significant increase in Fgl2 production, and this effect was abolished by using CC10 protein (Figures 5A,B) , indicating that CC10 directly modulates Fgl2 production in macrophages. In order to determine genes that were downregulated after stimulation by CC10 protein, we used DNA microarray analysis to screen for differentially expressed genes.", "In order to determine genes that were downregulated after stimulation by CC10 protein, we used DNA microarray analysis to screen for differentially expressed genes. THP-1 cells were cultured and PMA was added to induce differentiation into macrophages. The production of Fgl2 was stimulated by IFNγ.", "The production of Fgl2 was stimulated by IFNγ. The experimental group was treated with CC10 protein for microarray detection of differentially expressed genes. The results showed that the most obviously downregulated genes were UBE2W, HECTD1, MIR612, ATRX, SOX4, HBP1, and Fgl2 (Supplementary Table 1) .", "The results showed that the most obviously downregulated genes were UBE2W, HECTD1, MIR612, ATRX, SOX4, HBP1, and Fgl2 (Supplementary Table 1) . And then these genes were tested by qPCR. However, UBE2W, HECTD1, MIR612, ATRX, and SOX4 was not differentially expressed by qPCR, while HBP1 and fgl2 were still down-regulated genes.", "However, UBE2W, HECTD1, MIR612, ATRX, and SOX4 was not differentially expressed by qPCR, while HBP1 and fgl2 were still down-regulated genes. DNA microarray analysis identified HBP1 as a down-regulated gene involved in the pathological processes of the regulation of CC10. Recently, very limited studies have explored the role of HBP1 in FH.", "Recently, very limited studies have explored the role of HBP1 in FH. Nevertheless, the mechanistic functions of HBP1 in FH remain largely unexplored. Therefore, we selected this gene for further study.", "Therefore, we selected this gene for further study. qPCR analysis confirmed that mRNA levels of HBP1 were significantly decreased in THP-1 cells after CC10\n\nprotein stimulation compared to that in the PBS control group (Figure 6A ). We knocked down HBP1 using HBP1-siRNA. Then, transfection of HBP1-SiRNA into HUVECs was detected by qPCR and western-blotting methods.", "Then, transfection of HBP1-SiRNA into HUVECs was detected by qPCR and western-blotting methods. As expected, HBP1 knockdown led to significantly decreased expression of HBP1 (Figures 6B,C) . Furthermore, HBP1 knockdown impaired expression of Fgl2 (Figure 6D ), suggesting that HBP1 was able to activate Fgl2. HBP1-SiRNA was used to transfect HUVECs.", "HBP1-SiRNA was used to transfect HUVECs. Then, IFN-γ was added to induce the expression of Fgl2 followed by stimulation with CC10 protein (150 ng/ml) after 2 h. Finally, we explored the expression of Fgl2 by qPCR. The results showed that HBP1-SiRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in HUVECs (Figure 7) .", "The results showed that HBP1-SiRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in HUVECs (Figure 7) . That is to say, CC10 could suppress Fgl2 expression in macrophages. Such an effect may be mediated by the transcription factor HBP1. It is well-known that CC10 protein can suppress the immune response.", "It is well-known that CC10 protein can suppress the immune response. In animal models of allergic diseases of the respiratory tract, most of evidences confirm this inhibition (26) . Its function in FH has not been investigated yet.", "Its function in FH has not been investigated yet. Here, we used a murine FH model established by MHV-3 infection to explore the effects of CC10 in this disease process. To determine the role of CC10 in the pathogenesis of FH, CC10 protein was injected into a mouse FH model established by MHV-3 infection.", "To determine the role of CC10 in the pathogenesis of FH, CC10 protein was injected into a mouse FH model established by MHV-3 infection. MHV-3-induced liver injury in CC10-treated mice occurred rarely and the areas of lesions were much fewer than those in saline-treated control mice. In summary, these results suggested that CC10 could reduce pathological liver damage in this FH model together with lower mortality rates followed by MHV-3 infection.", "In summary, these results suggested that CC10 could reduce pathological liver damage in this FH model together with lower mortality rates followed by MHV-3 infection. MHV-3 induced fulminant viral hepatitis progresses rapidly and infected mice die within 3-5 days. Previous studies suggested fgl2 played a vital role in this process with a 15-40% increase of survival when fgl2 was deleted (12, 15, 27, 28) .", "Previous studies suggested fgl2 played a vital role in this process with a 15-40% increase of survival when fgl2 was deleted (12, 15, 27, 28) . Multiple inflammatory factors or mediators including TNF-α and IFN-γ, IL-1β and C5aR have been demonstrated to promote FH progression with significant discrepancies between liver damage and survival rate (29) (30) (31) (32) , which is accordant with our observation that CC10 substantially alleviated liver injury though survival rate improved mildly. The survival rate based on hours may be more accurate to examine the effect of CC10 on FH.", "The survival rate based on hours may be more accurate to examine the effect of CC10 on FH. It is speculated that fgl2 can mediate lethality in MHV-3-induced FH. This is due to the fact that fgl2 induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity (15) .", "This is due to the fact that fgl2 induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity (15) . To determine whether the tissue necrosis was mediated by Fgl2 in CC10-treated mice following infection, Fgl2 expression was observed. Results suggested that the expression of Fgl2 was significantly increased in MHV-3-induced FH mice and CC10 treatment significantly reduced the production of Fgl2 in the infected liver and serum.", "Results suggested that the expression of Fgl2 was significantly increased in MHV-3-induced FH mice and CC10 treatment significantly reduced the production of Fgl2 in the infected liver and serum. In addition, decreased fibrinogen deposition was also observed in the livers of CC10-treated mice. Therefore, our research results strongly clarify that the lower mortality of CC10-treated mice after MHV-3 infection is due to the lower levels of Fgl2 and decreased fibrinogen deposition.", "Therefore, our research results strongly clarify that the lower mortality of CC10-treated mice after MHV-3 infection is due to the lower levels of Fgl2 and decreased fibrinogen deposition. Indeed, it has been reported that Fgl2 is expressed on macrophages, and the expression of Fgl2 is believed to be induced by IFN-γ and TNF-α (22) . Cultured THP-1 cells activated by IFN-γ or IL-2 have been demonstrated, with induction of Fgl2 expression and enhanced activation of human prothrombin (23) .", "Cultured THP-1 cells activated by IFN-γ or IL-2 have been demonstrated, with induction of Fgl2 expression and enhanced activation of human prothrombin (23) . Therefore, in this study, we explored this cell line to investigate the modulation of CC10 on Fgl2. Surprisingly, we found that CC10 directly inhibited IFN-γ-induced Fgl2 expression in THP-1 cells.", "Surprisingly, we found that CC10 directly inhibited IFN-γ-induced Fgl2 expression in THP-1 cells. As we know, IFN-γ has proved to be the main cytokine that leads to the development and progression of FH. Also, it was shown that IFN-γ might exert its own proinflammatory biological function through enhancing Fgl2 expression.", "Also, it was shown that IFN-γ might exert its own proinflammatory biological function through enhancing Fgl2 expression. Therefore, in our study, CC10 might counter the effect of IFN-γ in the setting of FH, which substantiates its role in FH. These results demonstrated that CC10 regulates the expression of Fgl2 in macrophages.", "These results demonstrated that CC10 regulates the expression of Fgl2 in macrophages. In the current study, we used co-immunoprecipitation to analyze binding between CC10 and Fgl2. In this study, we investigated possible protein-protein interactions between CC10 and Fgl2 in vitro. The Chinese hamster ovary (CHO) cells transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2.", "The Chinese hamster ovary (CHO) cells transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2. Cellular proteins were immunoprecipitated with anti-CC10 antibody or anti-Fgl2 antibody. Immunoblotting was performed with anti-Fgl2 and anti-CC10 antibodies.", "Immunoblotting was performed with anti-Fgl2 and anti-CC10 antibodies. Immunoprecipitation of protein extracts from pcDNA 3.1-CC10 and pcDNA3.1-Fgl2 co-transfected CHO cells with anti-Fgl2 or anti-CC10 antibody followed by western blotting with Fgl2 and CC10 antibodies indicated that CC10 did not co-immunoprecipitate with Fgl2, showing that there is no direct relationship between CC10 and Fgl2 (data not shown). The results showed that CC10 has no direct interaction with Fgl2.", "The results showed that CC10 has no direct interaction with Fgl2. From our previous study the gene of fgl2 contributed profoundly in MHV-3 induced fulminant hepatitis and is extensively expressed in macrophages and endothelium (12, 33) . Our microarray indicated a CC10 down-regulated fgl2 expression and this is further confirmed by qPCR and Western blotting in vivo (peritoneal macrophages) and in vitro (THP-1, macrophage cell line).", "Our microarray indicated a CC10 down-regulated fgl2 expression and this is further confirmed by qPCR and Western blotting in vivo (peritoneal macrophages) and in vitro (THP-1, macrophage cell line). Therefore, it is reasonable to focus on macrophages to display the effect of CC10 on fgl2 expression and eventually mice survival. We entirely agree there may be other possibilities for a protective effect of CC10 to contribute to the disease process.", "We entirely agree there may be other possibilities for a protective effect of CC10 to contribute to the disease process. This is worth further studies. The potential receptor of CC10 has not been revealed yet. Our previous study have demonstrated that CC10 have effect of dendritic cells in allergic rhinitis (34) .", "Our previous study have demonstrated that CC10 have effect of dendritic cells in allergic rhinitis (34) . In this research, we evaluated the effect of CC10 on macrophages functions and found Fgl2 was substantially down-regulated upon CC10 treatment, therefore, we speculate that potential CC10 receptor may be also expressed on macrophages. The potential target of CC10 on other immune cells cannot be excluded.", "The potential target of CC10 on other immune cells cannot be excluded. DNA microarray analysis is one of the most powerful approaches for the potential identification of unexpected genes involved in pathogenic processes. By using this approach, HMGbox transcription factor 1 (HBP1) was found to be one of the most downregulated genes after CC10 treatment of THP-1 cells.", "By using this approach, HMGbox transcription factor 1 (HBP1) was found to be one of the most downregulated genes after CC10 treatment of THP-1 cells. HBP1 is a well-described transcriptional repressor that modulates expression of genes involved in cell cycle progression. In a recent study, it was found that HBP1 is a direct target of miR-21 and confirmed that HBP1 modulates the inhibitory function of miR-21-ASO in hepatosteatosis and carcinogenesis simultaneously (23) .", "In a recent study, it was found that HBP1 is a direct target of miR-21 and confirmed that HBP1 modulates the inhibitory function of miR-21-ASO in hepatosteatosis and carcinogenesis simultaneously (23) . HBP1 is an endogenous inhibitor of the Wnt signaling pathway in both normal and cancer cells. The tumor suppressor role of HBP1 has been reported in some malignancies, such as oral cancer and glioma (35) .", "The tumor suppressor role of HBP1 has been reported in some malignancies, such as oral cancer and glioma (35) . However, an association between HBP1 and Fgl2 has not been investigated yet. The current study clearly demonstrated that CC10 protects against MHV-3 induced FH via suppression of Fgl2 expression. Such effects might be mediated by HBP1.", "Such effects might be mediated by HBP1. However, the functional status of HBP1 in the CC10 pathway requires further research, and such studies are conducting in our laboratory. In conclusion, we demonstrated that CC10 could limit the immunopathological damage in MHV-3-induced FH mice.", "In conclusion, we demonstrated that CC10 could limit the immunopathological damage in MHV-3-induced FH mice. Our results suggest that enhancing CC10 expression by an immunotherapeutic approach might be an effective treatment for FH. HY performed all the described experiments and wrote the manuscript. YL assisted with some experiments, analyzed experimental results, and edited the manuscript.", "YL assisted with some experiments, analyzed experimental results, and edited the manuscript. HW analyzed experimental results. XW reviewed and edited the manuscript. JH, WY, DX, XL, GS, and QN provided experimental help and design." ]

[ "IMPORTANCE: Health care workers exposed to coronavirus disease 2019 (COVID-19) could be psychologically stressed. OBJECTIVE: To assess the magnitude of mental health outcomes and associated factors among health care workers treating patients exposed to COVID-19 in China. DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China.", "DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China. Health care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 were eligible. MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively.", "MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes. RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%.", "RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%. A total of 813 (64.7%) were aged 26 to 40 years, and 964 (76.7%) were women. Of all participants, 764 (60.8%) were nurses, and 493 (39.2%) were physicians; 760 (60.5%) worked in hospitals in Wuhan, and 522 (41.5%) were frontline health care workers.", "Of all participants, 764 (60.8%) were nurses, and 493 (39.2%) were physicians; 760 (60.5%) worked in hospitals in Wuhan, and 522 (41.5%) were frontline health care workers. A considerable proportion of participants reported symptoms of depression (634 [50.4%]), anxiety (560 [44.6%]), insomnia (427 [34.0%]), and distress (899 [71.5%]). Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers (eg, median [IQR] Patient Health Questionnaire scores among physicians vs nurses: 4.0 [1.0-7.0] vs 5.0 [2.0-8.0]; P = .007; median [interquartile range {IQR}] Generalized Anxiety Disorder scale scores among men vs women: 2.0 [0-6.0] vs 4.0 [1.0-7.0]; P < .001; median [IQR] Insomnia Severity Index scores among frontline vs second-line workers: 6.0 [2.0-11.0] vs 4.0 [1.0-8.0]; P < .001; median [IQR] Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 [8.5-34.5] vs 18.0 [6.0-28.0] in Hubei outside Wuhan and 15.0 [4.0-26.0] outside Hubei; P < .001).", "Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers (eg, median [IQR] Patient Health Questionnaire scores among physicians vs nurses: 4.0 [1.0-7.0] vs 5.0 [2.0-8.0]; P = .007; median [interquartile range {IQR}] Generalized Anxiety Disorder scale scores among men vs women: 2.0 [0-6.0] vs 4.0 [1.0-7.0]; P < .001; median [IQR] Insomnia Severity Index scores among frontline vs second-line workers: 6.0 [2.0-11.0] vs 4.0 [1.0-8.0]; P < .001; median [IQR] Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 [8.5-34.5] vs 18.0 [6.0-28.0] in Hubei outside Wuhan and 15.0 [4.0-26.0] outside Hubei; P < .001). Multivariable logistic regression analysis showed participants from outside Hubei province were associated with lower risk of experiencing symptoms of distress compared with those in Wuhan (odds ratio [OR], 0.62; 95% CI, 0.43-0.88; P = .008). Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression (OR, 1.52; 95% CI, 1.11-2.09; P = .01), anxiety (OR, 1.57; 95% CI, 1.22-2.02; P < .001), insomnia (OR, 2.97; 95% CI, 1.92-4.60; P < .001), and distress (OR, 1.60; 95% CI, 1.25-2.04; P < .001).", "Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression (OR, 1.52; 95% CI, 1.11-2.09; P = .01), anxiety (OR, 1.57; 95% CI, 1.22-2.02; P < .001), insomnia (OR, 2.97; 95% CI, 1.92-4.60; P < .001), and distress (OR, 1.60; 95% CI, 1.25-2.04; P < .001). CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19. Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range.", "Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range. Hyperarousal, median (IQR) 6.0(2.0, 10.0) 6.0(2.0, 9.0) .29" ]

[ "IMPORTANCE: Health care workers exposed to coronavirus disease 2019 (COVID-19) could be psychologically stressed. OBJECTIVE: To assess the magnitude of mental health outcomes and associated factors among health care workers treating patients exposed to COVID-19 in China. DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China.", "DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China. Health care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 were eligible. MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively.", "MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes. RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%.", "RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%. A total of 813 (64.7%) were aged 26 to 40 years, and 964 (76.7%) were women. Of all participants, 764 (60.8%) were nurses, and 493 (39.2%) were physicians; 760 (60.5%) worked in hospitals in Wuhan, and 522 (41.5%) were frontline health care workers.", "Of all participants, 764 (60.8%) were nurses, and 493 (39.2%) were physicians; 760 (60.5%) worked in hospitals in Wuhan, and 522 (41.5%) were frontline health care workers. A considerable proportion of participants reported symptoms of depression (634 [50.4%]), anxiety (560 [44.6%]), insomnia (427 [34.0%]), and distress (899 [71.5%]). Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers (eg, median [IQR] Patient Health Questionnaire scores among physicians vs nurses: 4.0 [1.0-7.0] vs 5.0 [2.0-8.0]; P = .007; median [interquartile range {IQR}] Generalized Anxiety Disorder scale scores among men vs women: 2.0 [0-6.0] vs 4.0 [1.0-7.0]; P < .001; median [IQR] Insomnia Severity Index scores among frontline vs second-line workers: 6.0 [2.0-11.0] vs 4.0 [1.0-8.0]; P < .001; median [IQR] Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 [8.5-34.5] vs 18.0 [6.0-28.0] in Hubei outside Wuhan and 15.0 [4.0-26.0] outside Hubei; P < .001).", "Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers (eg, median [IQR] Patient Health Questionnaire scores among physicians vs nurses: 4.0 [1.0-7.0] vs 5.0 [2.0-8.0]; P = .007; median [interquartile range {IQR}] Generalized Anxiety Disorder scale scores among men vs women: 2.0 [0-6.0] vs 4.0 [1.0-7.0]; P < .001; median [IQR] Insomnia Severity Index scores among frontline vs second-line workers: 6.0 [2.0-11.0] vs 4.0 [1.0-8.0]; P < .001; median [IQR] Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 [8.5-34.5] vs 18.0 [6.0-28.0] in Hubei outside Wuhan and 15.0 [4.0-26.0] outside Hubei; P < .001). Multivariable logistic regression analysis showed participants from outside Hubei province were associated with lower risk of experiencing symptoms of distress compared with those in Wuhan (odds ratio [OR], 0.62; 95% CI, 0.43-0.88; P = .008). Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression (OR, 1.52; 95% CI, 1.11-2.09; P = .01), anxiety (OR, 1.57; 95% CI, 1.22-2.02; P < .001), insomnia (OR, 2.97; 95% CI, 1.92-4.60; P < .001), and distress (OR, 1.60; 95% CI, 1.25-2.04; P < .001).", "Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression (OR, 1.52; 95% CI, 1.11-2.09; P = .01), anxiety (OR, 1.57; 95% CI, 1.22-2.02; P < .001), insomnia (OR, 2.97; 95% CI, 1.92-4.60; P < .001), and distress (OR, 1.60; 95% CI, 1.25-2.04; P < .001). CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19. Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range.", "Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range. Hyperarousal, median (IQR) 6.0(2.0, 10.0) 6.0(2.0, 9.0) .29" ]

[ "IMPORTANCE: Health care workers exposed to coronavirus disease 2019 (COVID-19) could be psychologically stressed. OBJECTIVE: To assess the magnitude of mental health outcomes and associated factors among health care workers treating patients exposed to COVID-19 in China. DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China.", "DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China. Health care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 were eligible. MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively.", "MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes. RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%.", "RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%. A total of 813 (64.7%) were aged 26 to 40 years, and 964 (76.7%) were women. Of all participants, 764 (60.8%) were nurses, and 493 (39.2%) were physicians; 760 (60.5%) worked in hospitals in Wuhan, and 522 (41.5%) were frontline health care workers.", "Of all participants, 764 (60.8%) were nurses, and 493 (39.2%) were physicians; 760 (60.5%) worked in hospitals in Wuhan, and 522 (41.5%) were frontline health care workers. A considerable proportion of participants reported symptoms of depression (634 [50.4%]), anxiety (560 [44.6%]), insomnia (427 [34.0%]), and distress (899 [71.5%]). Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers (eg, median [IQR] Patient Health Questionnaire scores among physicians vs nurses: 4.0 [1.0-7.0] vs 5.0 [2.0-8.0]; P = .007; median [interquartile range {IQR}] Generalized Anxiety Disorder scale scores among men vs women: 2.0 [0-6.0] vs 4.0 [1.0-7.0]; P < .001; median [IQR] Insomnia Severity Index scores among frontline vs second-line workers: 6.0 [2.0-11.0] vs 4.0 [1.0-8.0]; P < .001; median [IQR] Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 [8.5-34.5] vs 18.0 [6.0-28.0] in Hubei outside Wuhan and 15.0 [4.0-26.0] outside Hubei; P < .001).", "Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers (eg, median [IQR] Patient Health Questionnaire scores among physicians vs nurses: 4.0 [1.0-7.0] vs 5.0 [2.0-8.0]; P = .007; median [interquartile range {IQR}] Generalized Anxiety Disorder scale scores among men vs women: 2.0 [0-6.0] vs 4.0 [1.0-7.0]; P < .001; median [IQR] Insomnia Severity Index scores among frontline vs second-line workers: 6.0 [2.0-11.0] vs 4.0 [1.0-8.0]; P < .001; median [IQR] Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 [8.5-34.5] vs 18.0 [6.0-28.0] in Hubei outside Wuhan and 15.0 [4.0-26.0] outside Hubei; P < .001). Multivariable logistic regression analysis showed participants from outside Hubei province were associated with lower risk of experiencing symptoms of distress compared with those in Wuhan (odds ratio [OR], 0.62; 95% CI, 0.43-0.88; P = .008). Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression (OR, 1.52; 95% CI, 1.11-2.09; P = .01), anxiety (OR, 1.57; 95% CI, 1.22-2.02; P < .001), insomnia (OR, 2.97; 95% CI, 1.92-4.60; P < .001), and distress (OR, 1.60; 95% CI, 1.25-2.04; P < .001).", "Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression (OR, 1.52; 95% CI, 1.11-2.09; P = .01), anxiety (OR, 1.57; 95% CI, 1.22-2.02; P < .001), insomnia (OR, 2.97; 95% CI, 1.92-4.60; P < .001), and distress (OR, 1.60; 95% CI, 1.25-2.04; P < .001). CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19. Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range.", "Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range. Hyperarousal, median (IQR) 6.0(2.0, 10.0) 6.0(2.0, 9.0) .29" ]

[ "IMPORTANCE: Health care workers exposed to coronavirus disease 2019 (COVID-19) could be psychologically stressed. OBJECTIVE: To assess the magnitude of mental health outcomes and associated factors among health care workers treating patients exposed to COVID-19 in China. DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China.", "DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China. Health care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 were eligible. MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively.", "MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes. RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%.", "RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%. A total of 813 (64.7%) were aged 26 to 40 years, and 964 (76.7%) were women. Of all participants, 764 (60.8%) were nurses, and 493 (39.2%) were physicians; 760 (60.5%) worked in hospitals in Wuhan, and 522 (41.5%) were frontline health care workers.", "Of all participants, 764 (60.8%) were nurses, and 493 (39.2%) were physicians; 760 (60.5%) worked in hospitals in Wuhan, and 522 (41.5%) were frontline health care workers. A considerable proportion of participants reported symptoms of depression (634 [50.4%]), anxiety (560 [44.6%]), insomnia (427 [34.0%]), and distress (899 [71.5%]). Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers (eg, median [IQR] Patient Health Questionnaire scores among physicians vs nurses: 4.0 [1.0-7.0] vs 5.0 [2.0-8.0]; P = .007; median [interquartile range {IQR}] Generalized Anxiety Disorder scale scores among men vs women: 2.0 [0-6.0] vs 4.0 [1.0-7.0]; P < .001; median [IQR] Insomnia Severity Index scores among frontline vs second-line workers: 6.0 [2.0-11.0] vs 4.0 [1.0-8.0]; P < .001; median [IQR] Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 [8.5-34.5] vs 18.0 [6.0-28.0] in Hubei outside Wuhan and 15.0 [4.0-26.0] outside Hubei; P < .001).", "Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers (eg, median [IQR] Patient Health Questionnaire scores among physicians vs nurses: 4.0 [1.0-7.0] vs 5.0 [2.0-8.0]; P = .007; median [interquartile range {IQR}] Generalized Anxiety Disorder scale scores among men vs women: 2.0 [0-6.0] vs 4.0 [1.0-7.0]; P < .001; median [IQR] Insomnia Severity Index scores among frontline vs second-line workers: 6.0 [2.0-11.0] vs 4.0 [1.0-8.0]; P < .001; median [IQR] Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 [8.5-34.5] vs 18.0 [6.0-28.0] in Hubei outside Wuhan and 15.0 [4.0-26.0] outside Hubei; P < .001). Multivariable logistic regression analysis showed participants from outside Hubei province were associated with lower risk of experiencing symptoms of distress compared with those in Wuhan (odds ratio [OR], 0.62; 95% CI, 0.43-0.88; P = .008). Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression (OR, 1.52; 95% CI, 1.11-2.09; P = .01), anxiety (OR, 1.57; 95% CI, 1.22-2.02; P < .001), insomnia (OR, 2.97; 95% CI, 1.92-4.60; P < .001), and distress (OR, 1.60; 95% CI, 1.25-2.04; P < .001).", "Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression (OR, 1.52; 95% CI, 1.11-2.09; P = .01), anxiety (OR, 1.57; 95% CI, 1.22-2.02; P < .001), insomnia (OR, 2.97; 95% CI, 1.92-4.60; P < .001), and distress (OR, 1.60; 95% CI, 1.25-2.04; P < .001). CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19. Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range.", "Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range. Hyperarousal, median (IQR) 6.0(2.0, 10.0) 6.0(2.0, 9.0) .29" ]

[ "IMPORTANCE: Health care workers exposed to coronavirus disease 2019 (COVID-19) could be psychologically stressed. OBJECTIVE: To assess the magnitude of mental health outcomes and associated factors among health care workers treating patients exposed to COVID-19 in China. DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China.", "DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China. Health care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 were eligible. MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively.", "MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes. RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%.", "RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%. A total of 813 (64.7%) were aged 26 to 40 years, and 964 (76.7%) were women. Of all participants, 764 (60.8%) were nurses, and 493 (39.2%) were physicians; 760 (60.5%) worked in hospitals in Wuhan, and 522 (41.5%) were frontline health care workers.", "Of all participants, 764 (60.8%) were nurses, and 493 (39.2%) were physicians; 760 (60.5%) worked in hospitals in Wuhan, and 522 (41.5%) were frontline health care workers. A considerable proportion of participants reported symptoms of depression (634 [50.4%]), anxiety (560 [44.6%]), insomnia (427 [34.0%]), and distress (899 [71.5%]). Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers (eg, median [IQR] Patient Health Questionnaire scores among physicians vs nurses: 4.0 [1.0-7.0] vs 5.0 [2.0-8.0]; P = .007; median [interquartile range {IQR}] Generalized Anxiety Disorder scale scores among men vs women: 2.0 [0-6.0] vs 4.0 [1.0-7.0]; P < .001; median [IQR] Insomnia Severity Index scores among frontline vs second-line workers: 6.0 [2.0-11.0] vs 4.0 [1.0-8.0]; P < .001; median [IQR] Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 [8.5-34.5] vs 18.0 [6.0-28.0] in Hubei outside Wuhan and 15.0 [4.0-26.0] outside Hubei; P < .001).", "Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers (eg, median [IQR] Patient Health Questionnaire scores among physicians vs nurses: 4.0 [1.0-7.0] vs 5.0 [2.0-8.0]; P = .007; median [interquartile range {IQR}] Generalized Anxiety Disorder scale scores among men vs women: 2.0 [0-6.0] vs 4.0 [1.0-7.0]; P < .001; median [IQR] Insomnia Severity Index scores among frontline vs second-line workers: 6.0 [2.0-11.0] vs 4.0 [1.0-8.0]; P < .001; median [IQR] Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 [8.5-34.5] vs 18.0 [6.0-28.0] in Hubei outside Wuhan and 15.0 [4.0-26.0] outside Hubei; P < .001). Multivariable logistic regression analysis showed participants from outside Hubei province were associated with lower risk of experiencing symptoms of distress compared with those in Wuhan (odds ratio [OR], 0.62; 95% CI, 0.43-0.88; P = .008). Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression (OR, 1.52; 95% CI, 1.11-2.09; P = .01), anxiety (OR, 1.57; 95% CI, 1.22-2.02; P < .001), insomnia (OR, 2.97; 95% CI, 1.92-4.60; P < .001), and distress (OR, 1.60; 95% CI, 1.25-2.04; P < .001).", "Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression (OR, 1.52; 95% CI, 1.11-2.09; P = .01), anxiety (OR, 1.57; 95% CI, 1.22-2.02; P < .001), insomnia (OR, 2.97; 95% CI, 1.92-4.60; P < .001), and distress (OR, 1.60; 95% CI, 1.25-2.04; P < .001). CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19. Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range.", "Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range. Hyperarousal, median (IQR) 6.0(2.0, 10.0) 6.0(2.0, 9.0) .29" ]

[ "IMPORTANCE: Health care workers exposed to coronavirus disease 2019 (COVID-19) could be psychologically stressed. OBJECTIVE: To assess the magnitude of mental health outcomes and associated factors among health care workers treating patients exposed to COVID-19 in China. DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China.", "DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China. Health care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 were eligible. MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively.", "MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes. RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%.", "RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%. A total of 813 (64.7%) were aged 26 to 40 years, and 964 (76.7%) were women. Of all participants, 764 (60.8%) were nurses, and 493 (39.2%) were physicians; 760 (60.5%) worked in hospitals in Wuhan, and 522 (41.5%) were frontline health care workers.", "Of all participants, 764 (60.8%) were nurses, and 493 (39.2%) were physicians; 760 (60.5%) worked in hospitals in Wuhan, and 522 (41.5%) were frontline health care workers. A considerable proportion of participants reported symptoms of depression (634 [50.4%]), anxiety (560 [44.6%]), insomnia (427 [34.0%]), and distress (899 [71.5%]). Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers (eg, median [IQR] Patient Health Questionnaire scores among physicians vs nurses: 4.0 [1.0-7.0] vs 5.0 [2.0-8.0]; P = .007; median [interquartile range {IQR}] Generalized Anxiety Disorder scale scores among men vs women: 2.0 [0-6.0] vs 4.0 [1.0-7.0]; P < .001; median [IQR] Insomnia Severity Index scores among frontline vs second-line workers: 6.0 [2.0-11.0] vs 4.0 [1.0-8.0]; P < .001; median [IQR] Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 [8.5-34.5] vs 18.0 [6.0-28.0] in Hubei outside Wuhan and 15.0 [4.0-26.0] outside Hubei; P < .001).", "Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers (eg, median [IQR] Patient Health Questionnaire scores among physicians vs nurses: 4.0 [1.0-7.0] vs 5.0 [2.0-8.0]; P = .007; median [interquartile range {IQR}] Generalized Anxiety Disorder scale scores among men vs women: 2.0 [0-6.0] vs 4.0 [1.0-7.0]; P < .001; median [IQR] Insomnia Severity Index scores among frontline vs second-line workers: 6.0 [2.0-11.0] vs 4.0 [1.0-8.0]; P < .001; median [IQR] Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 [8.5-34.5] vs 18.0 [6.0-28.0] in Hubei outside Wuhan and 15.0 [4.0-26.0] outside Hubei; P < .001). Multivariable logistic regression analysis showed participants from outside Hubei province were associated with lower risk of experiencing symptoms of distress compared with those in Wuhan (odds ratio [OR], 0.62; 95% CI, 0.43-0.88; P = .008). Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression (OR, 1.52; 95% CI, 1.11-2.09; P = .01), anxiety (OR, 1.57; 95% CI, 1.22-2.02; P < .001), insomnia (OR, 2.97; 95% CI, 1.92-4.60; P < .001), and distress (OR, 1.60; 95% CI, 1.25-2.04; P < .001).", "Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression (OR, 1.52; 95% CI, 1.11-2.09; P = .01), anxiety (OR, 1.57; 95% CI, 1.22-2.02; P < .001), insomnia (OR, 2.97; 95% CI, 1.92-4.60; P < .001), and distress (OR, 1.60; 95% CI, 1.25-2.04; P < .001). CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19. Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range.", "Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range. Hyperarousal, median (IQR) 6.0(2.0, 10.0) 6.0(2.0, 9.0) .29" ]

[ "IMPORTANCE: Health care workers exposed to coronavirus disease 2019 (COVID-19) could be psychologically stressed. OBJECTIVE: To assess the magnitude of mental health outcomes and associated factors among health care workers treating patients exposed to COVID-19 in China. DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China.", "DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China. Health care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 were eligible. MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively.", "MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes. RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%.", "RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%. A total of 813 (64.7%) were aged 26 to 40 years, and 964 (76.7%) were women. Of all participants, 764 (60.8%) were nurses, and 493 (39.2%) were physicians; 760 (60.5%) worked in hospitals in Wuhan, and 522 (41.5%) were frontline health care workers.", "Of all participants, 764 (60.8%) were nurses, and 493 (39.2%) were physicians; 760 (60.5%) worked in hospitals in Wuhan, and 522 (41.5%) were frontline health care workers. A considerable proportion of participants reported symptoms of depression (634 [50.4%]), anxiety (560 [44.6%]), insomnia (427 [34.0%]), and distress (899 [71.5%]). Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers (eg, median [IQR] Patient Health Questionnaire scores among physicians vs nurses: 4.0 [1.0-7.0] vs 5.0 [2.0-8.0]; P = .007; median [interquartile range {IQR}] Generalized Anxiety Disorder scale scores among men vs women: 2.0 [0-6.0] vs 4.0 [1.0-7.0]; P < .001; median [IQR] Insomnia Severity Index scores among frontline vs second-line workers: 6.0 [2.0-11.0] vs 4.0 [1.0-8.0]; P < .001; median [IQR] Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 [8.5-34.5] vs 18.0 [6.0-28.0] in Hubei outside Wuhan and 15.0 [4.0-26.0] outside Hubei; P < .001).", "Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers (eg, median [IQR] Patient Health Questionnaire scores among physicians vs nurses: 4.0 [1.0-7.0] vs 5.0 [2.0-8.0]; P = .007; median [interquartile range {IQR}] Generalized Anxiety Disorder scale scores among men vs women: 2.0 [0-6.0] vs 4.0 [1.0-7.0]; P < .001; median [IQR] Insomnia Severity Index scores among frontline vs second-line workers: 6.0 [2.0-11.0] vs 4.0 [1.0-8.0]; P < .001; median [IQR] Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 [8.5-34.5] vs 18.0 [6.0-28.0] in Hubei outside Wuhan and 15.0 [4.0-26.0] outside Hubei; P < .001). Multivariable logistic regression analysis showed participants from outside Hubei province were associated with lower risk of experiencing symptoms of distress compared with those in Wuhan (odds ratio [OR], 0.62; 95% CI, 0.43-0.88; P = .008). Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression (OR, 1.52; 95% CI, 1.11-2.09; P = .01), anxiety (OR, 1.57; 95% CI, 1.22-2.02; P < .001), insomnia (OR, 2.97; 95% CI, 1.92-4.60; P < .001), and distress (OR, 1.60; 95% CI, 1.25-2.04; P < .001).", "Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression (OR, 1.52; 95% CI, 1.11-2.09; P = .01), anxiety (OR, 1.57; 95% CI, 1.22-2.02; P < .001), insomnia (OR, 2.97; 95% CI, 1.92-4.60; P < .001), and distress (OR, 1.60; 95% CI, 1.25-2.04; P < .001). CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19. Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range.", "Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range. Hyperarousal, median (IQR) 6.0(2.0, 10.0) 6.0(2.0, 9.0) .29" ]

[ "IMPORTANCE: Health care workers exposed to coronavirus disease 2019 (COVID-19) could be psychologically stressed. OBJECTIVE: To assess the magnitude of mental health outcomes and associated factors among health care workers treating patients exposed to COVID-19 in China. DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China.", "DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China. Health care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 were eligible. MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively.", "MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes. RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%.", "RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%. A total of 813 (64.7%) were aged 26 to 40 years, and 964 (76.7%) were women. Of all participants, 764 (60.8%) were nurses, and 493 (39.2%) were physicians; 760 (60.5%) worked in hospitals in Wuhan, and 522 (41.5%) were frontline health care workers.", "Of all participants, 764 (60.8%) were nurses, and 493 (39.2%) were physicians; 760 (60.5%) worked in hospitals in Wuhan, and 522 (41.5%) were frontline health care workers. A considerable proportion of participants reported symptoms of depression (634 [50.4%]), anxiety (560 [44.6%]), insomnia (427 [34.0%]), and distress (899 [71.5%]). Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers (eg, median [IQR] Patient Health Questionnaire scores among physicians vs nurses: 4.0 [1.0-7.0] vs 5.0 [2.0-8.0]; P = .007; median [interquartile range {IQR}] Generalized Anxiety Disorder scale scores among men vs women: 2.0 [0-6.0] vs 4.0 [1.0-7.0]; P < .001; median [IQR] Insomnia Severity Index scores among frontline vs second-line workers: 6.0 [2.0-11.0] vs 4.0 [1.0-8.0]; P < .001; median [IQR] Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 [8.5-34.5] vs 18.0 [6.0-28.0] in Hubei outside Wuhan and 15.0 [4.0-26.0] outside Hubei; P < .001).", "Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers (eg, median [IQR] Patient Health Questionnaire scores among physicians vs nurses: 4.0 [1.0-7.0] vs 5.0 [2.0-8.0]; P = .007; median [interquartile range {IQR}] Generalized Anxiety Disorder scale scores among men vs women: 2.0 [0-6.0] vs 4.0 [1.0-7.0]; P < .001; median [IQR] Insomnia Severity Index scores among frontline vs second-line workers: 6.0 [2.0-11.0] vs 4.0 [1.0-8.0]; P < .001; median [IQR] Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 [8.5-34.5] vs 18.0 [6.0-28.0] in Hubei outside Wuhan and 15.0 [4.0-26.0] outside Hubei; P < .001). Multivariable logistic regression analysis showed participants from outside Hubei province were associated with lower risk of experiencing symptoms of distress compared with those in Wuhan (odds ratio [OR], 0.62; 95% CI, 0.43-0.88; P = .008). Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression (OR, 1.52; 95% CI, 1.11-2.09; P = .01), anxiety (OR, 1.57; 95% CI, 1.22-2.02; P < .001), insomnia (OR, 2.97; 95% CI, 1.92-4.60; P < .001), and distress (OR, 1.60; 95% CI, 1.25-2.04; P < .001).", "Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression (OR, 1.52; 95% CI, 1.11-2.09; P = .01), anxiety (OR, 1.57; 95% CI, 1.22-2.02; P < .001), insomnia (OR, 2.97; 95% CI, 1.92-4.60; P < .001), and distress (OR, 1.60; 95% CI, 1.25-2.04; P < .001). CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19. Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range.", "Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range. Hyperarousal, median (IQR) 6.0(2.0, 10.0) 6.0(2.0, 9.0) .29" ]

[ "IMPORTANCE: Health care workers exposed to coronavirus disease 2019 (COVID-19) could be psychologically stressed. OBJECTIVE: To assess the magnitude of mental health outcomes and associated factors among health care workers treating patients exposed to COVID-19 in China. DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China.", "DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China. Health care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 were eligible. MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively.", "MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes. RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%.", "RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%. A total of 813 (64.7%) were aged 26 to 40 years, and 964 (76.7%) were women. Of all participants, 764 (60.8%) were nurses, and 493 (39.2%) were physicians; 760 (60.5%) worked in hospitals in Wuhan, and 522 (41.5%) were frontline health care workers.", "Of all participants, 764 (60.8%) were nurses, and 493 (39.2%) were physicians; 760 (60.5%) worked in hospitals in Wuhan, and 522 (41.5%) were frontline health care workers. A considerable proportion of participants reported symptoms of depression (634 [50.4%]), anxiety (560 [44.6%]), insomnia (427 [34.0%]), and distress (899 [71.5%]). Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers (eg, median [IQR] Patient Health Questionnaire scores among physicians vs nurses: 4.0 [1.0-7.0] vs 5.0 [2.0-8.0]; P = .007; median [interquartile range {IQR}] Generalized Anxiety Disorder scale scores among men vs women: 2.0 [0-6.0] vs 4.0 [1.0-7.0]; P < .001; median [IQR] Insomnia Severity Index scores among frontline vs second-line workers: 6.0 [2.0-11.0] vs 4.0 [1.0-8.0]; P < .001; median [IQR] Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 [8.5-34.5] vs 18.0 [6.0-28.0] in Hubei outside Wuhan and 15.0 [4.0-26.0] outside Hubei; P < .001).", "Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers (eg, median [IQR] Patient Health Questionnaire scores among physicians vs nurses: 4.0 [1.0-7.0] vs 5.0 [2.0-8.0]; P = .007; median [interquartile range {IQR}] Generalized Anxiety Disorder scale scores among men vs women: 2.0 [0-6.0] vs 4.0 [1.0-7.0]; P < .001; median [IQR] Insomnia Severity Index scores among frontline vs second-line workers: 6.0 [2.0-11.0] vs 4.0 [1.0-8.0]; P < .001; median [IQR] Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 [8.5-34.5] vs 18.0 [6.0-28.0] in Hubei outside Wuhan and 15.0 [4.0-26.0] outside Hubei; P < .001). Multivariable logistic regression analysis showed participants from outside Hubei province were associated with lower risk of experiencing symptoms of distress compared with those in Wuhan (odds ratio [OR], 0.62; 95% CI, 0.43-0.88; P = .008). Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression (OR, 1.52; 95% CI, 1.11-2.09; P = .01), anxiety (OR, 1.57; 95% CI, 1.22-2.02; P < .001), insomnia (OR, 2.97; 95% CI, 1.92-4.60; P < .001), and distress (OR, 1.60; 95% CI, 1.25-2.04; P < .001).", "Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression (OR, 1.52; 95% CI, 1.11-2.09; P = .01), anxiety (OR, 1.57; 95% CI, 1.22-2.02; P < .001), insomnia (OR, 2.97; 95% CI, 1.92-4.60; P < .001), and distress (OR, 1.60; 95% CI, 1.25-2.04; P < .001). CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19. Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range.", "Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range. Hyperarousal, median (IQR) 6.0(2.0, 10.0) 6.0(2.0, 9.0) .29" ]

[ "IMPORTANCE: Health care workers exposed to coronavirus disease 2019 (COVID-19) could be psychologically stressed. OBJECTIVE: To assess the magnitude of mental health outcomes and associated factors among health care workers treating patients exposed to COVID-19 in China. DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China.", "DESIGN, SETTINGS, AND PARTICIPANTS: This cross-sectional, survey-based, region-stratified study collected demographic data and mental health measurements from 1257 health care workers in 34 hospitals from January 29, 2020, to February 3, 2020, in China. Health care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 were eligible. MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively.", "MAIN OUTCOMES AND MEASURES: The degree of symptoms of depression, anxiety, insomnia, and distress was assessed by the Chinese versions of the 9-item Patient Health Questionnaire, the 7-item Generalized Anxiety Disorder scale, the 7-item Insomnia Severity Index, and the 22-item Impact of Event Scale–Revised, respectively. Multivariable logistic regression analysis was performed to identify factors associated with mental health outcomes. RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%.", "RESULTS: A total of 1257 of 1830 contacted individuals completed the survey, with a participation rate of 68.7%. A total of 813 (64.7%) were aged 26 to 40 years, and 964 (76.7%) were women. Of all participants, 764 (60.8%) were nurses, and 493 (39.2%) were physicians; 760 (60.5%) worked in hospitals in Wuhan, and 522 (41.5%) were frontline health care workers.", "Of all participants, 764 (60.8%) were nurses, and 493 (39.2%) were physicians; 760 (60.5%) worked in hospitals in Wuhan, and 522 (41.5%) were frontline health care workers. A considerable proportion of participants reported symptoms of depression (634 [50.4%]), anxiety (560 [44.6%]), insomnia (427 [34.0%]), and distress (899 [71.5%]). Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers (eg, median [IQR] Patient Health Questionnaire scores among physicians vs nurses: 4.0 [1.0-7.0] vs 5.0 [2.0-8.0]; P = .007; median [interquartile range {IQR}] Generalized Anxiety Disorder scale scores among men vs women: 2.0 [0-6.0] vs 4.0 [1.0-7.0]; P < .001; median [IQR] Insomnia Severity Index scores among frontline vs second-line workers: 6.0 [2.0-11.0] vs 4.0 [1.0-8.0]; P < .001; median [IQR] Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 [8.5-34.5] vs 18.0 [6.0-28.0] in Hubei outside Wuhan and 15.0 [4.0-26.0] outside Hubei; P < .001).", "Nurses, women, frontline health care workers, and those working in Wuhan, China, reported more severe degrees of all measurements of mental health symptoms than other health care workers (eg, median [IQR] Patient Health Questionnaire scores among physicians vs nurses: 4.0 [1.0-7.0] vs 5.0 [2.0-8.0]; P = .007; median [interquartile range {IQR}] Generalized Anxiety Disorder scale scores among men vs women: 2.0 [0-6.0] vs 4.0 [1.0-7.0]; P < .001; median [IQR] Insomnia Severity Index scores among frontline vs second-line workers: 6.0 [2.0-11.0] vs 4.0 [1.0-8.0]; P < .001; median [IQR] Impact of Event Scale–Revised scores among those in Wuhan vs those in Hubei outside Wuhan and those outside Hubei: 21.0 [8.5-34.5] vs 18.0 [6.0-28.0] in Hubei outside Wuhan and 15.0 [4.0-26.0] outside Hubei; P < .001). Multivariable logistic regression analysis showed participants from outside Hubei province were associated with lower risk of experiencing symptoms of distress compared with those in Wuhan (odds ratio [OR], 0.62; 95% CI, 0.43-0.88; P = .008). Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression (OR, 1.52; 95% CI, 1.11-2.09; P = .01), anxiety (OR, 1.57; 95% CI, 1.22-2.02; P < .001), insomnia (OR, 2.97; 95% CI, 1.92-4.60; P < .001), and distress (OR, 1.60; 95% CI, 1.25-2.04; P < .001).", "Frontline health care workers engaged in direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of depression (OR, 1.52; 95% CI, 1.11-2.09; P = .01), anxiety (OR, 1.57; 95% CI, 1.22-2.02; P < .001), insomnia (OR, 2.97; 95% CI, 1.92-4.60; P < .001), and distress (OR, 1.60; 95% CI, 1.25-2.04; P < .001). CONCLUSIONS AND RELEVANCE: In this survey of heath care workers in hospitals equipped with fever clinics or wards for patients with COVID-19 in Wuhan and other regions in China, participants reported experiencing psychological burden, especially nurses, women, those in Wuhan, and frontline health care workers directly engaged in the diagnosis, treatment, and care for patients with COVID-19. Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range.", "Text: Abbreviation: PHQ-9, 9-item Patient Health Questionnaire; GAD-7, 7-item Generalized Anxiety Disorder; ISI, 7-item Insomnia Severity Index; IES-R, 22-item Impact of Event Abbreviation: IES-R, 22-item Impact of Event Scale-Revised; IQR, interquartile range. Hyperarousal, median (IQR) 6.0(2.0, 10.0) 6.0(2.0, 9.0) .29" ]

[ "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)." ]