Corsica is a mountainous French island in the north-western Mediterranean Sea. It is a rural area, where pig farming is a major economic activity. Although no acute respiratory outbreaks due to swine influenza A viruses (swIAVs) have ever been reported in this free-ranging pig breeding system, influenza A viruses (IAVs) could be circulating within this pig population. A serological study was conducted as a first approach to domestic pigs and wild boars. Serum samples from 543 pigs raised on 91 different farms were collected during the 2013-2014 slaughtering season, and 279 sera from wild boars were obtained over four hunting seasons (between 2009 and 2014). They were first analysed by ELISA and then IAV positive and doubtful sera were subjected to haemagglutination inhibition tests using antigens representative of the four major enzootic swIAV lineages in Europe, i.e. avian-like swine H1N1 (H1avN1), pandemic-like swine H1N1 (H1N1pdm), H1N2 and H3N2. According to the ELISA results, 26.4% (CI95%: 17.7-36.7%) of herds had at least one positive animal (positive or doubtful by ELISA) and 12.4% (CI95%: 7.8-19.8%) of the pigs tested positive. Using the test characteristics (sensitivity and specificity), the true seroprevalence among Corsican pigs was estimated to be 16.4% (95% CI: 9.9-26.3). Antibodies directed against two different viral lineages were identified: H1N1pdm (in 66.2% and 45.8% of the IAV positive pigs and farms respectively) and H1avN1 (15.0% and 20.8% respectively). Evidence of exposure to viruses from two distinct lineages were detected on a single farm but in two different animals. Among the wild boars, 1.4% (CI95%: 0.4-3.6%) tested positive by ELISA and antibodies against the same two viruses were detected. Altogether, these results suggest that swIAVs from at least two different lineages are circulating among Corsican pigs, i.e. the H1N1pdm virus, probably introduced during the 2009 pandemic, and the H1avN1 virus, which is the most frequent swIAV in Europe. The low frequency of positive results observed in the Corsican wild boars hunted suggests that they would not play a major role in IAV dispersion dynamics on the island.

Annual vaccination is the most effective way to prevent seasonal influenza illness. Instituto Butantan (IB) performed clinical studies with its 2013, 2014 and 2015 seasonal trivalent influenza vaccines (inactivated split-virion). Prospective cohort studies were carried out to describe the safety and immunogenicity of Instituto Butantan influenza vaccines, in healthy adults and elderly, from 2013 to 2015. Immediately after the informed consent was signed, participants underwent blood collection followed by vaccination. On study days 1, 2 and 3 post-vaccination participants were contacted by the staff to evaluate the occurrence of solicited (local and systemic) and non-solicited adverse reactions. On study day 21 (+7) subjects returned to the clinical site for final safety assessments and blood collection to evaluate post-vaccination immunogenicity. The immunogenicity analyses were performed by means of hemagglutination inhibition (HI) assay. The immunogenicity endpoints were: seroprotection (SPR) and seroconversion (SCR) rates and the geometric mean HI antibody titer ratio (GMTR). The 2013 study was conducted at the Centro de Referência para Imunobiológicos Especiais (CRIE) and at the Centro de Pesquisa Clínica do Instituto da Criança, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo while the 2014 and 2015 studies were conducted at CRIE. The vaccine composition followed the WHO recommendation for the Southern hemisphere seasonal influenza vaccine. Forty-seven healthy adults and 13 elderly participated in the 2013 study, 60 healthy adults and 60 elderly in the 2014 study, and 62 healthy adults and 57 elderly in the 2015 study. In the 2013, 2014 and 2015 studies, pain was the most frequent local adverse reaction and headache the most frequent systemic adverse reaction. All observed adverse reactions were classified as mild or moderate and none as severe. SPR >70% and SPR >60% were observed in adults and elderly, respectively, for the three vaccine viruses, in the 2013, 2014 and 2015 studies. SCR >40% was observed in adults, for the three vaccine viruses, only in the 2014 study and SCR >30% was observed in the elderly, for the three vaccine viruses, only in the 2013 and 2014 studies. GMTR >2.5 among adults, for the three vaccine viruses was only observed in the 2013 study and GMTR >2.0 was observed among elderly, for the three vaccine viruses, in the 2013, 2014 and 2015 studies. The 2013, 2014 and 2015 seasonal influenza vaccines produced by Instituto Butantan were safe and immunogenic according to the immunogenicity criteria defined by the European Medicines Agency (EMA).

Wild bird-origin influenza A viruses (IAVs or avian influenza) have led to sporadic outbreaks among domestic poultry in the United States (US) and Canada, resulting in economic losses through the implementation of costly containment practices and destruction of birds. We used evolutionary analyses of virus sequence data to determine that 78 H5 low pathogenic avian influenza viruses (LPAIVs) isolated from domestic poultry in the US and Canada during 2001-2017 resulted from 18 independent virus introductions from wild birds. Within the wild bird reservoir, the hemagglutinin gene segments of H5 LPAIVs exist primarily as two co-circulating genetic sublineages, and our findings suggest the H5 gene segments flow within each migratory bird flyway and among adjacent flyways, with limited exchange between the non-adjacent Atlantic and Pacific Flyways. Phylogeographic analyses provided evidence that IAVs from dabbling ducks and swans/geese contributed to emergence of viruses among domestic poultry. H5 LPAIVs isolated from commercial farm poultry (i.e. turkey) were descended from a single introduction typically remain a single genotype, whereas those from live bird markets sometimes led to multiple genotypes, reflecting the potential for reassortment with other IAVs circulating within live bird markets. H5 LPAIV introduced from wild birds to domestic poultry represent economic threats to the U.S. poultry industry, and our data suggest that such introductions have been sporadic, controlled effectively through production monitoring and a stamping-out policy, and are, therefore, unlikely to result in sustained detections in commercial poultry operations.IMPORTANCE Integration of viral genome sequencing into influenza surveillance for wild birds and domestic poultry can elucidate evolutionary pathways of economically costly poultry pathogens. Evolutionary analyses of H5 LPAIVs detected in domestic poultry in US and Canada during 2001-2017 suggest that these viruses originated from repeated introductions of IAVs from wild birds, followed by various degrees of reassortment. Reassortment was observed where biosecurity was low and there were opportunities for more than one virus to circulate existed (e.g. congregations of birds from different premises such as live bird markets). None of the H5 lineages identified were maintained long term in domestic poultry, suggesting that management strategies have been effective in minimizing the impacts of virus introductions on US poultry production.

MicroRNAs (miRNAs) are small non-coding RNAs that are crucial post-transcriptional regulator for host mRNAs. Recent studies indicate that miRNAs may modulate host response during RNA viruses infection. However, the role of miRNAs in immune response against H5N1 infection is not clearly understood. In this study, we show that expression of cellular miRNA, miR-324-5p was downregulated in A549 cells in response to infection with RNA viruses, H5N1, A/PR8/H1N1, and NDV and transfection with polyI:C. We found that miR-324-5p inhibited H5N1 replication by targeting the PB1 viral RNA of H5N1 in host cells. In addition, transcriptome analysis revealed that miR-324-5p enhanced the expression of Type-I, Type-III interferons, and interferons-inducible genes (ISGs) by targeting CUEDC2, the negative regulator of JAK1-STAT3 pathway. Altogether, these findings highlights that the miR-324-5p plays a crucial role in host defense against H5N1 by targeting viral PB1 and host CUEDC2 to inhibit H5N1 replication.IMPORTANCE Highly pathogenic influenza A virus (HPAIV) continues to pose a pandemic threat globally. From 2003-2017, H5N1 HPAI has caused 453 human deaths with a high mortality rate of 52.74%. This work shows that miR-324-5p suppresses the H5N1 HPAI viral replication by directly targeting viral genome (thereby inhibits viral gene expression) and cellular CUEDC2 gene, the negative regulator of interferon pathway (thereby enhance anti-viral genes). Our study enhance the knowledge of role of microRNAs in cellular response to viral infection. Also, the study provides help in understanding how the host cells utilizes small RNAs in controlling the viral burden.

Influenza viruses use distinct antibody escape mechanisms depending on the overall complexity of the antibody response that is encountered. When grown in the presence of a hemagglutinin (HA) monoclonal antibody, influenza viruses typically acquire a single HA mutation that reduces the binding of that specific monoclonal antibody. In contrast, when confronted with mixtures of HA monoclonal antibodies or polyclonal sera that have antibodies that bind several HA epitopes, influenza viruses acquire mutations that increase HA binding to host cells. Recent data from our laboratory and others suggest that some humans possess antibodies that are narrowly focused on HA epitopes that were present in influenza virus strains that they were likely exposed to in childhood. Here, we completed a series of experiments to determine if humans with narrowly focused HA antibody responses are able to select for influenza virus antigenic escape variants in ovo We identified three human donors that possessed HA antibody responses that were heavily focused on a single HA antigenic site. Sera from all three of these donors selected single HA escape mutations during in ovo passage experiments, similar to what has been previously reported for single monoclonal antibodies. These single HA mutations directly reduced binding of serum antibodies used for selection. We propose that new antigenic variants of influenza viruses might originate in individuals who produce antibodies that are narrowly focused on HA epitopes that were present in viral strains that they encountered in childhood.IMPORTANCE Influenza vaccine strains must be updated frequently since circulating viral strains continuously change in antigenically important epitopes. Our previous studies have demonstrated that some individuals possess antibody responses that are narrowly focused on epitopes that were present in viral strains that they encountered during childhood. Here, we show that influenza viruses rapidly escape this type of polyclonal antibody response when grown in ovo by acquiring single mutations that directly prevent antibody binding. These studies improve our understanding of how influenza viruses evolve when confronted with narrowly focused polyclonal human antibodies.

Using the Hemagglutinin (HA) protein peptide array of H1N1 pdm09 and a panel of swine antisera against various swine influenza H1 and H3 clusters, we identified three immunoreactive epitopes with one (peptide 15) located in HA1 (amino acids 57-71) and two (peptides 121 and 139) in HA2 (amino acids 481-495 and 553-566). Further analysis showed that all swine antisera of H1 clusters efficiently recognized two HA2 epitopes; peptides 121 and 139, with only a subset of antisera reactive to HA1-derived peptide 15. Interestingly, none of these peptides were reactive to SIV H3 antisera. Finally, intranasal inoculation of peptides 15 and 121 into pigs revealed that peptide 121, not peptide 15, was able to generate antibody responses in some animals. The results of our experiments provide an important foundation for further analyzing the immune response against these peptides during natural viral infection and also provide peptide substrates for diagnostic assays.

The nonstructural protein (NSs) of severe fever with thrombocytopenia syndrome phlebovirus (SFTSV) sequesters TANK-binding kinase 1 (TBK1) into NSs-induced cytoplasmic structures to inhibit the phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3) and subsequent interferon beta (IFN-β) production. Although the C-terminal region of SFTSV NSs (NSs66-249) has been linked to the formation of NSs-induced cytoplasmic structures and inhibition of host IFN-β responses, the role of the N-terminal region in antagonising host antiviral responses remains to be defined. Herein, we demonstrate that two conserved amino acids at positions 21 and 23 in the SFTSV and heartland virus (HRTV) NSs are essential for suppression of IRF3 phosphorylation and IFN-β mRNA expression following infection with SFTSV or recombinant influenza virus lacking the NS1 gene. Surprisingly, formation of SFTSV/HRTV NSs-induced cytoplasmic structures is not essential for inhibition of host antiviral responses. Rather, association between SFTSV/HRTV NSs and TBK1 is required for suppression of mitochondrial antiviral signalling protein (MAVS)-mediated activation of IFN-β promoter activity. Although the SFTSV NSs did not prevent ubiquitination of TBK1, they associate with TBK1 though its N-terminal kinase domain (residues 1-307) to block the autophosphorylation of TBK1. Further, we found that both wild-type and 21/23A mutant NSs of SFTSV suppressed the NLRP3 inflammasome-dependent IL-1β secretion, suggesting that the importance of these residues is restricted to TBK1-dependent IFN signalling. Together, our findings strongly implicate the two conserved amino acids at positions 21 and 23 of SFTSV/HRTV NSs in the inhibition of host interferon responses.ImportanceRecognition of viruses by host innate immune systems plays a critical role not only in providing resistance to viral infection, but also in initiation of antigen-specific adaptive immune responses against viruses. Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging infectious disease caused by the SFTS phlebovirus (SFTSV), a highly pathogenic tick-borne phlebovirus. The 294 amino acid nonstructural protein (NSs) of SFTSV associates with TANK-binding kinase 1 (TBK1), a key regulator of host innate antiviral immunity, to inhibit interferon beta (IFN-β) production and enhance viral replication. Herein, we demonstrate that two conserved amino acids at positions 21 and 23 in the NSs of SFTSV and heartland virus, another tick-borne phlebovirus, are essential for association with TBK1 and suppression of IFN-β production. Our results provide important insight into the molecular mechanisms by which SFTSV NSs helps to counteract host antiviral strategies.

In 2009, a pandemic H1N1 influenza A virus (IAV) (pH1N1) emerged in the human population from swine causing a pandemic. Importantly, this virus is still circulating in humans seasonally. To analyze the evolution of pH1N1 in humans, we sequenced viral genes encoding proteins inhibiting general gene expression (nonstructural protein 1 [NS1] and PA-X) from circulating seasonal viruses and compared them to the viruses isolated at the origin of the pandemic. Recent pH1N1 viruses contain amino acid changes in the NS1 protein (E55K, L90I, I123V, E125D, K131E, and N205S), as previously described (A. M. Clark, A. Nogales, L. Martinez-Sobrido, D. J. Topham, and M. L. DeDiego, J Virol 91:e00721-17, 2017, https://doi.org/10.1128/JVI.00721-17), and amino acid changes in the PA-X protein (V100I, N204S, R221Q, and L229S). These amino acid differences between early and more recent pH1N1 isolates are responsible for increased NS1-mediated inhibition of host gene expression and decreased PA-X-mediated shutoff, including innate immune response genes. In addition, currently circulating pH1N1 viruses have acquired amino acid changes in the PA protein (V100I, P224S, N321K, I330V, and R362K). A recombinant pH1N1 virus containing PA, PA-X, and NS1 genes from currently circulating viruses is fitter in replication in cultured cells and in mice and is slightly more pathogenic than the original ancestor pH1N1 virus. These results demonstrate the need to monitor the evolution of pH1N1 in humans for mutations in the viral genome that could result in enhanced virulence. Importantly, these results further support our previous findings suggesting that inhibition of global gene expression mediated by NS1 and PA-X proteins is subject to a balance which can determine virus pathogenesis and fitness.IMPORTANCE IAVs emerge in humans from animal reservoirs, causing unpredictable pandemics. One of these pandemics was caused by an H1N1 virus in 2009, and this virus is still circulating seasonally. To analyze host-virus adaptations likely affecting influenza virus pathogenesis, protein amino acid sequences from viruses circulating at the beginning of the pandemic and those circulating currently were compared. Currently circulating viruses have incorporated amino acid changes in two viral proteins (NS1 and PA-X), affecting innate immune responses, and in the PA gene. These amino acid differences led to increased NS1-mediated and decreased PA-X-mediated inhibition of host gene expression. A recombinant pH1N1 virus containing PA, PA-X, and NS1 genes from recently circulating viruses is fitter in replication in tissue culture cells and in mice, and the virus is more pathogenic in vivo Importantly, these results suggest that a balance in the abilities of NS1 and PA-X to induce host shutoff is beneficial for IAVs.