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The recognition of viral nucleic acids by host pattern recognition receptors (PRRs) is critical for initiating innate immune responses against viral infections. These innate immune responses are mediated by the induction of interferons (IFNs), IFN-stimulated genes (ISGs) and pro-inflammatory cytokines. However, regulatory mechanisms are critical to avoid excessive or long-lasting innate immune responses that may cause detrimental hyperinflammation. Here, we identified a novel regulatory function of the ISG, IFN alpha inducible protein 27 (IFI27) in counteracting the innate immune responses triggered by cytoplasmic RNA recognition and binding. Our model systems included three unrelated viral infections caused by Influenza A virus (IAV), Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), and Sendai virus (SeV), and transfection with an analog of double-stranded (ds) RNA. Furthermore, we found that IFI27 has a positive effect on IAV and SARS-CoV-2 replication, most likely due to its ability to counteract host-induced antiviral responses, including in vivo. We also show that IFI27 interacts with nucleic acids and PRR retinoic acid-inducible gene I (RIG-I), being the interaction of IFI27 with RIG-I most likely mediated through RNA binding. Interestingly, our results indicate that interaction of IFI27 with RIG-I impairs RIG-I activation, providing a molecular mechanism for the effect of IFI27 on modulating innate immune responses. Our study identifies a molecular mechanism that may explain the effect of IFI27 in counterbalancing innate immune responses to RNA viral infections and preventing excessive innate immune responses. Therefore, this study will have important implications in drug design to control viral infections and viral-induced pathology.

This work was supported by the Spanish Ministry of Science, Innovation and Universities (RTI-2018-094213-A-I00 and PID-2021- 123810OBI00) to MD, the “Atracció n de Talento Investigador” programme (2017-T1/BMD-5155) funded by the “Comunidad de Madrid” to MD, the Spanish National Research Council (CSIC)’s Global Health Platform (PTI Salud Global, CSIC-COV19-012/ 012202020E086) to MD, and a “Ramon y Cajal” Incorporation grant (RYC-2017) from the Spanish Ministry of Science, Innovation and Universities and research incorporation grant (202240I182) from CSIC to AN.

Background Coronaviruses usually cause mild respiratory disease in humans but as seen recently, some human coronaviruses can cause more severe diseases, such as the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the global spread of which has resulted in the ongoing coronavirus pandemic. Results In this study we analyzed the potential of using iron oxide nanoparticles (IONPs) coated with biocompatible molecules like dimercaptosuccinic acid (DMSA), 3-aminopropyl triethoxysilane (APS) or carboxydextran (FeraSpin™ R), as well as iron oxyhydroxide nanoparticles (IOHNPs) coated with sucrose (Venofer®), or iron salts (ferric ammonium citrate -FAC), to treat and/or prevent SARS-CoV-2 infection. At non-cytotoxic doses, IONPs and IOHNPs impaired virus replication and transcription, and the production of infectious viruses in vitro, either when the cells were treated prior to or after infection, although with different efficiencies. Moreover, our data suggest that SARS-CoV-2 infection affects the expression of genes involved in cellular iron metabolism. Furthermore, the treatment of cells with IONPs and IOHNPs affects oxidative stress and iron metabolism to different extents, likely influencing virus replication and production. Interestingly, some of the nanoparticles used in this work have already been approved for their use in humans as anti-anemic treatments, such as the IOHNP Venofer®, and as contrast agents for magnetic resonance imaging in small animals like mice, such as the FeraSpin™ R IONP. Conclusions Therefore, our results suggest that IONPs and IOHNPs may be repurposed to be used as prophylactic or therapeutic treatments in order to combat SARS-CoV-2 infection., This work was supported by the following Grants: CSIC-COV19-012/012202020E154 funded by the Spanish National Research Council Interdisciplinary Thematic Platform (PTI) Global Health (PTI Salud Global), SGL2103021 funded by the European Commission-NextGenerationEU (Regulation EU2020/2094) through CSIC’s Global Health Platform (PTI Salud Global); PDC2021-120759-100 funded by MCIN/AEI/10. 13039/50110 00110 33 and by the “European Union NextGenerationEU/PRTR”, PID2020-112685RB-100 funded by MCIN/AEI/10. 13039/50110 00110 33, and the “Atracción de Talento Investigador” programme (2017-T1/BMD-5155) funded by the “Comunidad de Madrid”. Y. Portilla was first a predoctoral FPU scholar (FPU15/06170) funded by MCIN/AEI/10. 13039/50110 00110 33 and by “ESF Investing in your future”, then a predoctoral scholar funded by CSIC-COV19-012/012202020E154 and is now a postdoctoral scholar funded by the European Commission-NextGenerationEU (Regulation EU2020/2094) through the CSIC’s Global Health Platform (PTI Salud Global, SGL2103021). D. López-García received a JAE-INTRO 2020 Fellowship from the Spanish National Research Council (CSIC, JAEINT-20-01805). V. Mulens-Arias was a postdoctoral scholar working under a Juan de La Cierva-Incorporación Contract (IJCI-2017-31447) funded by MCIN/AEI/10. 13039/50110 00110 33. N. Daviu is a predoctoral scholar (FPU18/04828) funded by MCIN/AEI/10. 13039/50110 00110 33 and by “ESF Investing in your future”. This research work was performed in the framework of the Nanomedicine CSIC HUB (ref. 202180E048).

17 Pág., PA-X is a nonstructural protein of influenza A virus (IAV), which is encoded by the polymerase acidic (PA) N-terminal region that contains a C-terminal +1 frameshifted sequence. IAV PA-X protein modulates virus-induced host innate immune responses and viral pathogenicity via suppression of host gene expression or cellular shutoff, through cellular mRNA cleavage. Highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype naturally infect different avian species, they have an enormous economic impact in the poultry farming, and they also have zoonotic and pandemic potential, representing a risk to human public health. In the present study, we describe a novel bacterium-based approach to identify amino acid residues in the PA-X protein of the HPAIV A/Viet Nam/1203/2004 H5N1 that are important for its ability to inhibit host protein expression or cellular shutoff activity. Identified PA-X mutants displayed a reduced shutoff activity compared to that of the wild-type A/Viet Nam/1203/2004 H5N1 PA-X protein. Notably, this new bacterium-based screening allowed us to identify amino acid residues widely distributed over the entire N-terminal region of PA-X. Furthermore, we found that some of the residues affecting A/Viet Nam/1203/2004 H5N1 PA-X host shutoff activity also affect PA polymerase activity in a minigenome assay. This information could be used for the rational design of new and more effective compounds with antiviral activity against IAV. Moreover, our results demonstrate the feasibility of using this bacterium-based approach to identify amino acid residues important for the activity of viral proteins to inhibit host gene expression. IMPORTANCE Highly pathogenic avian influenza viruses continue to pose a huge threat to global animal and human health. Despite of the limited genome size of Influenza A virus (IAV), the virus encodes eight main viral structural proteins and multiple accessory nonstructural proteins, depending on the IAV type, subtype, or strain. One of the IAV accessory proteins, PA-X, is encoded by the polymerase acidic (PA) protein and is involved in pathogenicity through the modulation of IAV-induced host inflammatory and innate immune responses. However, the molecular mechanism(s) of IAV PA-X regulation of the host immune response is not well understood. Here, we used, for the first time, a bacterium-based approach for the identification of amino acids important for the ability of IAV PA-X to induce host shutoff activity and describe novel residues relevant for its ability to inhibit host gene expression, and their contribution in PA polymerase activity., This study was supported with funds from Comunidad de Madrid (Spain), reference 2017-T1/BMD-5155, and the Spanish Ministry of Science, Innovation, and Universities (RTI-2018-094213-A-I00) to M.L.D. and from a Ramon y Cajal Incorporation grant (RYC-2017) from the Spanish Ministry of Science, Innovation, and Universities to A.N. D.L.-G. received a JAE-INTRO 2020 fellowship from the Spanish National Research Council (CSIC; JAEINT-20-01805). This research was partially funded by the New York Influenza Center of Excellence (NYICE), a member of the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Department of Health and Human Services, Centers of Excellence for Influenza Research and Surveillance (CEIRS) contract HHSN272201400005C (NYICE).

Influenza A viruses (IAVs) infect a broad range of hosts, including multiple avian and mammalian species. The frequent emergence of novel IAV strains in different hosts, including in humans, results in the need for vigilance and ongoing development of new approaches to fighting or prevent those infections. Canine influenza is a contagious respiratory disease in dogs caused by two subtypes of IAV, the equine-origin H3N8 canine influenza virus (CIV), and the avian-origin H3N2 CIV. A novel approach to influenza vaccination involves single-cycle infectious influenza A viruses (sciIAVs), which are defective for an essential viral gene. They are propagated in complementing cell lines which provide the missing gene in trans. As sciIAV cannot complete their replication cycle in regular cells they are limited to a single round of viral replication. Because of their safety profile and ability to express foreign antigens inside infected cells, sciIAVs have served both as live-attenuated vaccines and as vaccine vectors for the expression of heterologous antigens. Here, we describe experimental procedures for the generation of a single-cycle infectious CIV (sciCIV), where the viral hemagglutinin (HA) gene was exchanged for the gene for green fluorescent protein (GFP). Complementation of the viral HA protein is provided in trans by stable HA-expressing cell lines. Methods for the in vitro characterization of HA deficient but GFP-expressing sciCIV (sciCIV ΔHA/GFP) are described, as well as its use as a potential vaccine.

Influenza A viruses (IAVs) represent a serious concern globally because they are capable of rapid spread and cause severe disease in humans and other animals. The development and implementation of plasmid-based reverse genetics approaches have allowed the manipulation and recovery of recombinant IAVs from complementary DNA copies of the viral genome. Furthermore, IAV reverse genetics have provided researchers an efficient and powerful platform to introduce specific changes in the viral genome with the final goal of studying IAV biology, designing more effective vaccine strategies, and to reduce the rates of incidence and mortality associated with viral infections. In this review, we briefly discuss IAV reverse genetics and their applications to prevent IAV infections., Influenza research in LM-S laboratory has been partially funded by the New York Influenza Center of Excellence (NYICE, NIH 272201400005C), a member of the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Department of Health and Human Services, Centers of Excellence for Influenza Research and Surveillance (CEIRS) contract No. HHSN272201400005C (NYICE), the NIH/NIAID 1R01AI145332, and the Department of Defense (DoD) Peer Reviewed Medical Research Program (PRMRP) grant W81XWH-18-1-0460. Research in M.L.D laboratory was supported with funds from Comunidad de Madrid, Spain, reference 2017-T1/BMD-5155 and the Spanish Ministry of Science, Innovation and Universities (RTI-2018-094213-A-I00). Research in A.N laboratory was partially funded with the ‘Ramon y Cajal’ Fellowship RYC-2017 from Spanish Ministry of Economy, Industry and Competitiveness and the Spanish Ministry of Science, Innovation and Universities (AGL2017-82570-R).

Influenza A viruses (IAV) spread rapidly and can infect a broad range of avian or mammalian species, having a tremendous impact in human and animal health and the global economy. IAV have evolved to develop efficient mechanisms to counteract innate immune responses, the first host mechanism that restricts IAV infection and replication. One key player in this fight against host-induced innate immune responses is the IAV non-structural 1 (NS1) protein that modulates antiviral responses and virus pathogenicity during infection. In the last decades, the implementation of reverse genetics approaches has allowed to modify the viral genome to design recombinant IAV, providing researchers a powerful platform to develop effective vaccine strategies. Among them, different levels of truncation or deletion of the NS1 protein of multiple IAV strains has resulted in attenuated viruses able to induce robust innate and adaptive immune responses, and high levels of protection against wild-type (WT) forms of IAV in multiple animal species and humans. Moreover, this strategy allows the development of novel assays to distinguish between vaccinated and/or infected animals, also known as Differentiating Infected from Vaccinated Animals (DIVA) strategy. In this review, we briefly discuss the potential of NS1 deficient or truncated IAV as safe, immunogenic and protective live-attenuated influenza vaccines (LAIV) to prevent disease caused by this important animal and human pathogen.

227-256 pp, Influenza A viruses (IAVs) infect a broad range of hosts, including multiple avian and mammalian species. The frequent emergence of novel IAV strains in different hosts, including in humans, results in the need for vigilance and ongoing development of new approaches to fighting or prevent those infections. Canine influenza is a contagious respiratory disease in dogs caused by two subtypes of IAV, the equine-origin H3N8 canine influenza virus (CIV), and the avian-origin H3N2 CIV. A novel approach to influenza vaccination involves single-cycle infectious influenza A viruses (sciIAVs), which are defective for an essential viral gene. They are propagated in complementing cell lines which provide the missing gene in trans. As sciIAV cannot complete their replication cycle in regular cells they are limited to a single round of viral replication. Because of their safety profile and ability to express foreign antigens inside infected cells, sciIAVs have served both as live-attenuated vaccines and as vaccine vectors for the expression of heterologous antigens. Here, we describe experimental procedures for the generation of a single-cycle infectious CIV (sciCIV), where the viral hemagglutinin (HA) gene was exchanged for the gene for green fluorescent protein (GFP). Complementation of the viral HA protein is provided in trans by stable HA-expressing cell lines. Methods for the in vitro characterization of HA deficient but GFP-expressing sciCIV (sciCIV ΔHA/GFP) are described, as well as its use as a potential vaccine., This research was partially funded by the New York Influenza Center of Excellence (NYICE), a member of the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Department of Health and Human Services, Centers of Excellence for Influenza Research and Surveillance (CEIRS) contract No. HHSN272201400005C (NYICE). This research was partially funded by a “Ramon y Cajal” Incorporation grant (RYC-2017) from the Spanish Ministry of Science, Innovation and Universities to A.N.

SARS-CoV-2 uses the angiotensin-converting enzyme 2 (ACE2) and neuropilin-1 (NRP1) receptors for entry into cells, and the serine protease TMPRSS2 for S protein priming. Inhibition of protease activity or the engagement with ACE2 and NRP1 receptors has been shown to be an effective strategy for blocking infectivity and viral spreading. Valproic acid (VPA; 2-propylpentanoic acid) is an epigenetic drug approved for clinical use. It produces potent antiviral and anti-inflammatory effects through its function as a histone deacetylase (HDAC) inhibitor. Here, we propose VPA as a potential candidate to tackle COVID-19, in which rapid viral spread and replication, and hyperinflammation are crucial elements.We used diverse cell lines (HK-2, Huh-7, HUVEC, Caco-2, and BEAS-2B) to analyze the effect of VPA and other HDAC inhibitors on the expression of the ACE-2 and NRP-1 receptors and their ability to inhibit infectivity, viral production, and the inflammatory response. Treatment with VPA significantly reduced expression of the ACE2 and NRP1 host proteins in all cell lines through a mechanism mediated by its HDAC inhibitory activity. The effect is maintained after SARS-CoV-2 infection. Consequently, the treatment of cells with VPA before infection impairs production of SARS-CoV-2 infectious viruses, but not that of other ACE2- and NRP1-independent viruses (VSV and HCoV-229E). Moreover, the addition of VPA 1 h post-infection with SARS-CoV-2 reduces the production of infectious viruses in a dose-dependent manner without significantly modifying the genomic and subgenomic messenger RNAs (gRNA and sg mRNAs) or protein levels of N protein. The production of inflammatory cytokines (TNF-α and IL-6) induced by TNF-α and SARS-CoV-2 infection is diminished in the presence of VPA.Our data showed that VPA blocks three essential processes determining the severity of COVID-19. It downregulates the expression of ACE2 and NRP1, reducing the infectivity of SARS-CoV-2; it decreases viral yields, probably because it affects virus budding or virions stability; and it dampens the triggered inflammatory response. Thus, administering VPA could be considered a safe treatment for COVID-19 patients until vaccines have been rolled out across the world.

25 Pág. Centro de Investigación en Sanidad Animal (CISA), Influenza A viruses (IAV) can infect a broad range of mammalian and avian species. However, the host innate immune system provides defenses that restrict IAV replication and infection. Likewise, IAV have evolved to develop efficient mechanisms to counteract host antiviral responses to efficiently replicate in their hosts. The IAV PA-X and NS1 non-structural proteins are key virulence factors that modulate innate immune responses and virus pathogenicity during infection. To study the determinants of IAV pathogenicity and their functional co-evolution, we evaluated amino acid differences in the PA-X and NS1 proteins of early (1996–1997) and more recent (since 2016) H5N1 IAV. H5N1 IAV have zoonotic and pandemic potential and represent an important challenge both in poultry farming and human health. The results indicate that amino acid changes occurred over time, affecting the ability of these two non-structural H5N1 IAV proteins to inhibit gene expression and affecting virus pathogenicity. These results highlight the importance to monitor the evolution of these two virulence factors of IAV, which could result in enhanced viral replication and virulence., This work was supported with funds from Comunidad de Madrid (Spain), reference 2017-T1/BMD-5155 and the Spanish Ministry of Science, Innovation and Universities (RTI-2018-094213-A-I00) to M.L.D and a “Ramon y Cajal” Incorporation grant (RYC-2017) from Spanish Ministry of Science, Innovation and Universities to A.N.

Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component. Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci (eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene), including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types., Graphical abstract, D’Antonio et al. characterize associations between GWAS signals for COVID-19 disease and eQTLs in 69 human tissues to identify causal variants and their underlying molecular mechanisms. They show that diverse symptoms and disease severity of COVID-19 are associated with variants affecting gene expression in a wide variety of tissues.

Centro de Investigación en Sanidad Animal (CISA), The influenza A virus (IAV) is able to infect multiple mammalian and avian species, and in humans IAV is responsible for annual seasonal epidemics and occasional pandemics of respiratory disease with significant health and economic impacts. Studying IAV involves laborious secondary methodologies to identify infected cells. Therefore, to circumvent this requirement, in recent years, multiple replication-competent infectious IAV expressing traceable reporter genes have been developed. These IAVs have been very useful for in vitro and/or in vivo studies of viral replication, identification of neutralizing antibodies or antivirals, and in studies to evaluate vaccine efficacy, among others. In this report, we describe, for the first time, the generation and characterization of two replication-competent influenza A/Puerto Rico/8/1934 H1N1 (PR8) viruses where the viral non-structural protein 1 (NS1) was substituted by the monomeric (m)Cherry fluorescent or the NanoLuc luciferase (Nluc) proteins. The ΔNS1 mCherry was able to replicate in cultured cells and in Signal Transducer and Activator of Transcription 1 (STAT1) deficient mice, although at a lower extent than a wild-type (WT) PR8 virus expressing the same mCherry fluorescent protein (WT mCherry). Notably, expression of either reporter gene (mCherry or Nluc) was detected in infected cells by fluorescent microscopy or luciferase plate readers, respectively. ΔNS1 IAV expressing reporter genes provide a novel approach to better understand the biology and pathogenesis of IAV, and represent an excellent tool to develop new therapeutic approaches against IAV infections., This research was partially funded by the New York Influenza Center of Excellence (NYICE) and the Center for Research on Influenza Pathogenesis (CRIP), two members of the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Department of Health and Human Services, Centers of Excellence for Influenza Research and Surveillance (CEIRS) contract No. HHSN272201400005C (NYICE) and HHSN272201400008C (CRIP), and a “Ramon y Cajal” Incorporation grant (RYC-2017) from Spanish Ministry of Science, Innovation and Universities to A.N, 16 Pág. This article belongs to the Section Animal Viruses

© 2020 by the authors, Influenza virus infections represent a serious public health problem causing contagious respiratory disease and substantial morbidity and mortality in humans, resulting in a considerable economic burden worldwide. Notably, the number of deaths due to influenza exceeds that of any other known pathogen. Moreover, influenza infections can differ in their intensity, from mild respiratory disease to pneumonia, which can lead to death. Articles in this Special Issue have addressed different aspects of influenza in human health, and the advances in influenza research leading to the development of better therapeutics and vaccination strategies, with a special focus on the study of factors associated with innate or adaptive immune responses to influenza vaccination and/or infection., This work was supported with funds from Comunidad de Madrid, Spain, reference 2017-T1/BMD-5155 and the Spanish Ministry of Science, Innovation and Universities (RTI-2018-094213-A-I00) to M.L.D and the “Ramon y Cajal” Fellowship RYC-2017 from Spanish Ministry of Economy, Industry and Competitiveness to A.N.

The continual emergence of novel influenza A strains from non-human hosts requires constant vigilance and the need for ongoing research to identify strains that may pose a human public health risk. Since 1999, canine H3 influenza A viruses (CIVs) have caused many thousands or millions of respiratory infections in dogs in the United States. While no human infections with CIVs have been reported to date, these viruses could pose a zoonotic risk. In these studies, the National Institutes of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) network collaboratively demonstrated that CIVs replicated in some primary human cells and transmitted effectively in mammalian models. While people born after 1970 had little or no pre-existing humoral immunity against CIVs, the viruses were sensitive to existing antivirals and we identified a panel of H3 cross-reactive human monoclonal antibodies (hmAbs) that could have prophylactic and/or therapeutic value. Our data predict these CIVs posed a low risk to humans. Importantly, we showed that the CEIRS network could work together to provide basic research information important for characterizing emerging influenza viruses, although there were valuable lessons learned., Author summary The 2009 influenza pandemic was a stark reminder that ongoing vigilance is critical to protect the public from an influenza pandemic. The continual evolution of influenza viruses and emergence from animal reservoirs, leads to the need to quickly identify strains that pose a public health risk. In these studies, members of the National Institutes of Allergy and Infectious Diseases (NIAID) Centers for Excellence in Influenza Research and Surveillance (CEIRS) network worked together to demonstrate that the emerging canine H3 influenza viruses posed a low risk to public health and identified several therapeutic options in the event of an emergence. In addition to providing important new basic research, many lessons were learned that may be important in dealing with any emerging disease outbreak.

© 2019 Nogales, Aydillo, Ávila-Pérez, Escalera, Chiem, Cadagan, DeDiego, Li, García-Sastre and Martínez-Sobrido., Influenza viruses are important pathogens that affect multiple animal species, including humans. There are four types of influenza viruses: A, B, C, and D (IAV, IBV, ICV, and IDV, respectively). IAV and IBV are currently circulating in humans and are responsible of seasonal epidemics (IAV and IBV) and occasional pandemics (IAV). ICV is known to cause mild infections in humans and pigs, while the recently identified IDV primarily affect cattle and pigs. Influenza non-structural protein 1 (NS1) is a multifunctional protein encoded by the NS segment in all influenza types. The main function of NS1 is to counteract the host antiviral defense, including the production of interferon (IFN) and IFN-stimulated genes (ISGs), and therefore is considered an important viral pathogenic factor. Despite of homologous functions, the NS1 protein from the diverse influenza types share little amino acid sequence identity, suggesting possible differences in their mechanism(s) of action, interaction(s) with host factors, and contribution to viral replication and/or pathogenesis. In addition, although the NS1 protein of IAV, IBV and, to some extent ICV, have been previously studied, it is unclear if IDV NS1 has similar properties. Using an approach that allow us to express NS1 independently of the nuclear export protein from the viral NS segment, we have generated recombinant IAV expressing IAV, IBV, ICV, and IDV NS1 proteins. Although recombinant viruses expressing heterotypic (IBV, ICV, and IDV) NS1 proteins were able to replicate similarly in canine MDCK cells, their viral fitness was impaired in human A549 cells and they were highly attenuated in vivo. Our data suggest that despite the similarities to effectively counteract innate immune responses in vitro, the NS1 proteins of IBV, ICV, or IDV do not fully complement the functions of IAV NS1, resulting in deficient viral replication and pathogenesis in vivo., This research was partially funded by the New York Influenza Center of Excellence (NYICE), a member of the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Department of Health and Human Services, Centers of Excellence for Influenza Research and Surveillance (CEIRS) Contract No. HHSN272201400005C (NYICE); by the Department of Defense (DoD) Peer Reviewed Medical Research Program (PRMRP) grant W81XWH-18-1-0460 to LM-S; by the NIH R01AI141889 to FL; and by the Center for Research in Influenza Pathogenesis (CRIP, CEIRS Contract No. HHSN272201400008C to AG-S).

Centro de Investigación en Sanidad Animal (CISA), The influenza A virus (IAV) PA-X protein is a virulence factor that selectively degrades host mRNAs leading to protein shutoff. This function modulates host inflammation, antiviral responses, cell apoptosis, and pathogenesis. In this work we describe a novel approach based on the use of bacteria and plasmid encoding of the PA-X gene under the control of the bacteriophage T7 promoter to identify amino acid residues important for A/Brevig Mission/1/1918 H1N1 PA-X's shutoff activity. Using this system, we have identified PA-X mutants encoding single or double amino acid changes, which diminish its host shutoff activity, as well as its ability to counteract interferon responses upon viral infection. This novel bacteria-based approach could be used for the identification of viral proteins that inhibit host gene expression as well as the amino acid residues responsible for inhibition of host gene expression., This research was funded by the Spanish Ministry of Science, Innovation and Universities (RTI-2018-094213-A-I00) to M.L.D. and a “Ramon y Cajal” Incorporation grant (RYC-2017) from the Spanish Ministry of Science, Innovation and Universities to A.N. This research was partially funded by the New York Influenza Center of Excellence (NYICE), a member of the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Department of Health and Human Services, Centers of Excellence for Influenza Research and Surveillance (CEIRS) contract No. HHSN272201400005C (NYICE)., 17 Pág.

Influenza NS1 protein is the main viral protein counteracting host innate immune responses, allowing the virus to efficiently replicate in interferon (IFN)-competent systems. In this study, we analyzed NS1 protein variability within influenza A (IAV) H3N2 viruses infecting humans during the 2012-2013 season. We also evaluated the impact of the mutations on the ability of NS1 proteins to inhibit host innate immune responses and general gene expression. Surprisingly, a previously unidentified mutation in the double-stranded RNA (dsRNA)-binding domain (I64T) decreased NS1-mediated general inhibition of host protein synthesis by decreasing its interaction with cleavage and polyadenylation specificity factor 30 (CPSF30), leading to increased innate immune responses after viral infection. Notably, a recombinant A/Puerto Rico/8/34 H1N1 virus encoding the H3N2 NS1-T64 protein was highly attenuated in mice, most likely because of its ability to induce higher antiviral IFN responses at early times after infection and because this virus is highly sensitive to the IFN-induced antiviral state. Interestingly, using peripheral blood mononuclear cells (PBMCs) collected at the acute visit (2 to 3 days after infection), we show that the subject infected with the NS1-T64 attenuated virus has diminished responses to interferon and to interferon induction, suggesting why this subject could be infected with this highly IFN-sensitive virus. These data demonstrate the importance of influenza virus surveillance in identifying new mutations in the NS1 protein, affecting its ability to inhibit innate immune responses and, as a consequence, the pathogenicity of the virus. IMPORTANCE Influenza A and B viruses are one of the most common causes of respiratory infections in humans, causing 1 billion infections and between 300,000 and 500,000 deaths annually. Influenza virus surveillance to identify new mutations in the NS1 protein affecting innate immune responses and, as a consequence, the pathogenicity of the circulating viruses is highly relevant. Here, we analyzed amino acid variability in the NS1 proteins from human seasonal viruses and the effect of the mutations in innate immune responses and virus pathogenesis. A previously unidentified mutation in the dsRNA-binding domain decreased NS1-mediated general inhibition of host protein synthesis and the interaction of the protein with CPSF30. This mutation led to increased innate immune responses after viral infection, augmented IFN sensitivity, and virus attenuation in mice. Interestingly, using PBMCs, the subject infected with the virus encoding the attenuating mutation induced decreased antiviral responses, suggesting why this subject could be infected with this virus.

© The Author(s)., A large number of human genes associated with viral infections contain single nucleotide polymorphisms (SNPs), which represent a genetic variation caused by the change of a single nucleotide in the DNA sequence. SNPs are located in coding or non-coding genomic regions and can affect gene expression or protein function by different mechanisms. Furthermore, they have been linked to multiple human diseases, highlighting their medical relevance. Therefore, the identification and analysis of this kind of polymorphisms in the human genome has gained high importance in the research community, and an increasing number of studies have been published during the last years. As a consequence of this exhaustive exploration, an association between the presence of some specific SNPs and the susceptibility or severity of many infectious diseases in some risk population groups has been found. In this review, we discuss the relevance of SNPs that are important to understand the pathology derived from influenza A virus (IAV) infections in humans and the susceptibility of some individuals to suffer more severe symptoms. We also discuss the importance of SNPs for IAV vaccine effectiveness, This work was supported with funds from Comunidad de Madrid, Spain, reference 2017-T1/BMD-5155 and the Spanish Ministry of Science, Innovation and Universities (RTI-2018-094213-A-I00) to M.L.D and the “Ramon y Cajal” Fellowship RYC-2017 from Spanish Ministry of Economy, Industry and Competitiveness to A.N.

We describe a novel function for the interferon (IFN)-induced protein 44-like (IFI44L) gene in negatively modulating innate immune responses induced after virus infections. Furthermore, we show that decreasing IFI44L expression impairs virus production and that IFI44L expression negatively modulates the antiviral state induced by an analog of double-stranded RNA (dsRNA) or by IFN treatment. The mechanism likely involves the interaction of IFI44L with cellular FK506-binding protein 5 (FKBP5), which in turn interacts with kinases essential for type I and III IFN responses, such as inhibitor of nuclear factor kappa B (IκB) kinase alpha (IKKα), IKKβ, and IKKε. Consequently, binding of IFI44L to FKBP5 decreased interferon regulatory factor 3 (IRF-3)-mediated and nuclear factor kappa-B (NF-κB) inhibitor (IκBα)-mediated phosphorylation by IKKε and IKKβ, respectively. According to these results, IFI44L is a good target for treatment of diseases associated with excessive IFN levels and/or proinflammatory responses and for reduction of viral replication., This project was funded with federal funds from the National Institute of Allergy andInfectious Diseases (NIAID), National Institutes of Health (NIH), Department of Health and Human Services, under CEIRS contract HHSN272201400005C to D.J.T.; through a University of Rochester Research Award to M.L.D.; and with funds from Comunidad de Madrid, Madrid, Spain (reference 2017-T1/BMD-5155), to M.L.D.

Memory B cells (MBCs) are key determinants of the B cell response to influenza virus infection and vaccination, but the effect of different forms of influenza antigen exposure on MBC populations has received little attention. We analyzed peripheral blood mononuclear cells and plasma collected following human H3N2 influenza infection to investigate the relationship between hemagglutinin-specific antibody production and changes in the size and character of hemagglutinin-reactive MBC populations. Infection produced increased concentrations of plasma IgG reactive to the H3 head of the infecting virus, to the conserved stalk, and to a broad chronological range of H3s consistent with original antigenic sin responses. H3-reactive IgG MBC expansion after infection included reactivity to head and stalk domains. Notably, expansion of H3 head-reactive MBC populations was particularly broad and reflected original antigenic sin patterns of IgG production. Findings also suggest that early-life H3N2 infection “imprints” for strong H3 stalk-specific MBC expansion. Despite the breadth of MBC expansion, the MBC response included an increase in affinity for the H3 head of the infecting virus. Overall, our findings indicate that H3-reactive MBC expansion following H3N2 infection is consistent with maintenance of response patterns established early in life, but nevertheless includes MBC adaptation to the infecting virus. IMPORTANCE Rapid and vigorous virus-specific antibody responses to influenza virus infection and vaccination result from activation of preexisting virus-specific memory B cells (MBCs). Understanding the effects of different forms of influenza virus exposure on MBC populations is therefore an important guide to the development of effective immunization strategies. We demonstrate that exposure to the influenza hemagglutinin via natural infection enhances broad protection through expansion of hemagglutinin-reactive MBC populations that recognize head and stalk regions of the molecule. Notably, we show that hemagglutinin-reactive MBC expansion reflects imprinting by early-life infection and that this might apply to stalk-reactive, as well as to head-reactive, MBCs. Our findings provide experimental support for the role of MBCs in maintaining imprinting effects and suggest a mechanism by which imprinting might confer heterosubtypic protection against avian influenza viruses. It will be important to compare our findings to the situation after influenza vaccination.

Avian influenza vaccines exhibit poor immunogenicity in humans. We hypothesized that one factor underlying weak B cell responses was sequence divergence between avian and seasonal influenza hemagglutinin proteins, thus limiting the availability of adequate CD4 T cell help. To test this, a novel chimeric hemagglutinin protein (cH7/3) was derived, comprised of the stem domain from seasonal H3 hemagglutinin and the head domain from avian H7. Immunological memory to seasonal influenza was established in mice, through strategies that included seasonal inactivated vaccines, Flumist, and synthetic peptides derived from the H3 stalk domain. After establishment of memory, mice were vaccinated with H7 or cH7/3 protein. The cH7/3 Ag was able to recall H3-specific CD4 T cells, and this potentiated CD4 T cell response was associated with enhanced early germinal center response and rapid elicitation of Abs to H7, including Abs specific for the H7 head domain. These results suggest that in pandemic situations, inclusion of CD4 T cell epitopes from seasonal viruses have the potential to overcome the poor immunogenicity of avian vaccines by helping B cells and conferring greater subtype-specific Ab response to viral HA., This work was supported by National Institutes of Health Grant HHSN272201400005C (to A.J.S.), Leading Advanced Projects for medical innovation from the Japan Agency for Medical Research and Development (JP18am001007), and the National Institute of Allergy and Infectious Diseases–funded Center for Research on Influenza Pathogenesis under Grant HHSN272201400008C (to Y.K.). A.T.D. was supported in part by National Institutes of Health Grant 5T32AI007285.

Studying influenza A virus (IAV) requires the use of secondary approaches to detect the presence of virus in infected cells. To overcome this problem, we and others have generated recombinant IAV expressing fluorescent or luciferase reporter genes. These foreign reporter genes can be used as valid surrogates to track the presence of virus. However, the limited capacity for incorporating foreign sequences in the viral genome forced researchers to select a fluorescent or a luciferase reporter gene, depending on the type of study. To circumvent this limitation, we engineered a novel recombinant replication-competent bireporter IAV (BIRFLU) expressing both fluorescent and luciferase reporter genes. In cultured cells, BIRFLU displayed growth kinetics comparable to those of wild-type (WT) virus and was used to screen neutralizing antibodies or compounds with antiviral activity. The expression of two reporter genes allows monitoring of viral inhibition by fluorescence or bioluminescence, overcoming the limitations associated with the use of one reporter gene as a readout. In vivo, BIRFLU effectively infected mice, and both reporter genes were detected using in vivo imaging systems (IVIS). The ability to generate recombinant IAV harboring multiple foreign genes opens unique possibilities for studying virus-host interactions and for using IAV in high-throughput screenings (HTS) to identify novel antivirals that can be incorporated into the therapeutic armamentarium to control IAV infections. Moreover, the ability to genetically manipulate the viral genome to express two foreign genes offers the possibility of developing novel influenza vaccines and the feasibility for using recombinant IAV as vaccine vectors to treat other pathogen infections., This research was partially funded by the New York Influenza Center of Excellence (NYICE) (NIH 272201400005C), a member of National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), U.S. Department of Health and Human Services, Centers of Excellence for Influenza Research and Surveillance (CEIRS), contract no. HHSN272201400005C (NYICE), and by U.S. Department of Defense (DoD) Peer Reviewed Medical Research Program (PRMRP) grant W81XWH-18-1-0460 to L.M.-S.

Influenza A viruses (IAV) can infect a broad range of animal hosts, including humans. In humans, IAV causes seasonal annual epidemics and occasional pandemics, representing a serious public health and economic problem, which is most effectively prevented through vaccination. The defense mechanisms that the host innate immune system provides restrict IAV replication and infection. Consequently, to successfully replicate in interferon (IFN)-competent systems, IAV has to counteract host antiviral activities, mainly the production of IFN and the activities of IFN-induced host proteins that inhibit virus replication. The IAV multifunctional proteins PA-X and NS1 are virulence factors that modulate the innate immune response and virus pathogenicity. Notably, these two viral proteins have synergistic effects in the inhibition of host protein synthesis in infected cells, although using different mechanisms of action. Moreover, the control of innate immune responses by the IAV NS1 and PA-X proteins is subject to a balance that can determine virus pathogenesis and fitness, and recent evidence shows co-evolution of these proteins in seasonal viruses, indicating that they should be monitored for enhanced virulence. Importantly, inhibition of host gene expression by the influenza NS1 and/or PA-X proteins could be explored to develop improved live-attenuated influenza vaccines (LAIV) by modulating the ability of the virus to counteract antiviral host responses. Likewise, both viral proteins represent a reasonable target for the development of new antivirals for the control of IAV infections. In this review, we summarize the role of IAV NS1 and PA-X in controlling the antiviral response during viral infection., Influenza virus research in the Luis Mart í nez-Sobrido and David J. Topham laboratories is partially funded by the National Institute of Allergy and Infectious Diseases (NIAID), the National Institutes of Health (NIH), and the Department of Health and Human Services under the Centers of Excellence for Influenza Research and Surveillance (CEIRS) contract No. HHSN272201400005C (New York Influenza Center of Excellence, NYICE); by the Department of Defense (DoD) PR17046 (Luis Martinez-Sobrido); and with funds from a University of Rochester Research Award to Aitor Nogales and Marta Lopez de Diego. We also give thanks for the funds from Comunidad de Madrid, Spain, reference 2017-T1/BMD-5155 (Marta Lopez de Diego).

Summary Influenza vaccination does not provide 100% protection from infection, partly due to antigenic drift of the haemagglutinin (HA) protein. Low serum antibody titres increase the risk of infection. To determine whether there were additional correlates of risk, we examined the relationship between human serum immunity and antigenic variation in seasonal H3N2 influenza viruses. Seasonal H3N2 vaccine strains grown in the presence of heterogeneous human or mono‐specific ferret antisera selected variants with mutations in the HA antigenic sites. Surprisingly, circulating strains infecting human subjects in the same seasons displayed mutations in the same positions, although only in one case did the change correspond to the same amino acid. Serum antibody titres were lower against both the in vitro selected and clinical isolates compared with the vaccine strains, suggesting that the mutations are relevant to vaccine failure. Antibody titres were also significantly lower in sera from infected subjects than in non‐infected subjects, suggesting relatively poor responses to vaccination in the infected subjects. Collectively, the data suggest that risk from influenza infection is a result of poor response to vaccination, as well as encounter with drifted seasonal influenza virus antigenic variants. The results also show that directed selection under human immune pressure could reveal antigenic variants relevant to real‐world drifted viruses, helping in annual vaccine re‐formulation.

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.

31 Pág. Centro de Investigación en Sanidad Animal (CISA), This review discusses the human immune responses to influenza infection with some insights from studies using animal models, such as experimental infection of mice. Recent technological advances in the study of human immune responses have greatly added to our knowledge of the infection and immune responses, and therefore much of the focus is on recent studies that have moved the field forward. We consider the complexity of the adaptive response generated by many sequential encounters through infection and vaccination., This article has been made freely available online courtesy of TAUNS Laboratories.

Rapid changes in influenza A virus (IAV) antigenicity create challenges in surveillance, disease diagnosis, and vaccine development. Further, serological methods for studying antigenic properties of influenza viruses often rely on animal models and therefore may not fully reflect the dynamics of human immunity. We hypothesized that arrays of human monoclonal antibodies (hmAbs) to influenza could be employed in a pattern-recognition approach to expedite IAV serology, and to study the antigenic evolution of newly emerging viruses. Using the multiplex, label-free Arrayed Imaging Reflectometry (AIR) platform, we have demonstrated that such arrays readily discriminated among various subtypes of IAVs, including H1, H3 seasonal strains, and avian-sourced human H7 viruses. Array responses also allowed the first determination of antigenic relationships among IAV strains directly from hmAb responses. Finally, correlation analysis of antibody binding to all tested IAV subtypes allowed efficient identification of broadly reactive clones. In addition to specific applications in the context of understanding influenza biology with potential utility in “universal” flu vaccine development, these studies validate AIR as a platform technology for studying antigenic properties of viruses, and also antibody properties in a high-throughput manner. We further anticipate that this approach will facilitate advances in the study of other viral pathogens.

Influenza virus hemagglutinin (HA) and neuraminidase (NA) proteins elicit protective antibody responses and therefore, are used as targets for vaccination, especially the HA protein. However, these proteins are subject to antigenic drift, decreasing vaccine efficacy, and few to no studies have analyzed antigenic variability of these proteins by growing the viruses under immune pressure provided by human sera. In this work, we show that after growing different influenza virus strains under immune pressure, the selection of amino acid changes in the NA protein is much more limited than the selection in the HA protein, suggesting that the NA protein could remain more conserved under immune pressure. Interestingly, all the mutations in the HA and NA proteins affected protein antigenicity, and many of the selected amino acid changes were located at the same positions found in viruses circulating. These studies could help to inform HA and NA protein residues targeted by antibody responses after virus infection in humans and are very relevant to update the strains used for influenza virus vaccination each year and to improve the currently available vaccines.

Viroporins are viral proteins with ion channel (IC) activity that play an important role in several processes, including virus replication and pathogenesis. While many coronaviruses (CoVs) encode two viroporins, severe acute respiratory syndrome CoV (SARS-CoV) encodes three: proteins 3a, E, and 8a. Additionally, proteins 3a and E have a PDZ-binding motif (PBM), which can potentially bind over 400 cellular proteins which contain a PDZ domain, making them potentially important for the control of cell function. In the present work, a comparative study of the functional motifs included within the SARS-CoV viroporins was performed, mostly focusing on the roles of the IC and PBM of E and 3a proteins. Our results showed that the full-length E and 3a proteins were required for maximal SARS-CoV replication and virulence, whereas viroporin 8a had only a minor impact on these activities. A virus missing both the E and 3a proteins was not viable, whereas the presence of either protein with a functional PBM restored virus viability. E protein IC activity and the presence of its PBM were necessary for virulence in mice. In contrast, the presence or absence of the homologous motifs in protein 3a did not influence virus pathogenicity. Therefore, dominance of the IC and PBM of protein E over those of protein 3a was demonstrated in the induction of pathogenesis in mice., IMPORTANCE Collectively, these results demonstrate key roles for the ion channel and PBM domains in optimal virus replication and pathogenesis and suggest that the viral viroporins and PBMs are suitable targets for antiviral therapy and for mutation in attenuated SARS-CoV vaccines.

A number of investigators have suggested that exposure to low-dose radiation may pose a potentially serious health risk. However, the majority of these studies have focused on the short-term rather than long-term effects of exposure to fixed source radiation, and few have examined the effects of internal contamination. Additionally, very few studies have focused on exposure in juveniles, when organs are still developing and could be more sensitive to the toxic effects of radiation. To specifically address whether early-life radiation injury may affect long-term immune competence, we studied 14-day-old juvenile pups that were either 5 Gy total-body irradiated or injected internally with 50 μCi soluble (137)Cs, then infected with influenza A virus at 26 weeks after exposure. After influenza infection, all groups demonstrated immediate weight loss. We found that externally irradiated, infected animals failed to recover weight relative to age-matched infected controls, but internally (137)Cs contaminated and infected animals had a weight recovery with a similar rate and degree as controls. Externally and internally irradiated mice demonstrated reduced levels of club cell secretory protein (CCSP) message in their lungs after influenza infection. The externally irradiated group did not recover CCSP expression even at the two-week time point after infection. Although the antibody response and viral titers did not appear to be affected by either radiation modality, there was a slight increase in monocyte chemoattractant protein (MCP)-1 expression in the lungs of externally irradiated animals 14 days after influenza infection, with increased cellular infiltration present. Notably, an increase in the number of regulatory T cells was seen in the mediastinal lymph nodes of irradiated mice relative to uninfected mice. These data confirm the hypothesis that early-life irradiation may have long-term consequences on the immune system, leading to an altered antiviral response.

Antigenic drift of the hemagglutinin (HA) and neuraminidase (NA) influenza virus proteins contributes to reduced vaccine efficacy. To analyze antigenic drift in human seasonal H1N1 viruses derived from the 2009 pandemic H1N1 virus (pH1N1-like viruses) accounts for the limited effectiveness (around 40%) of vaccination against pH1N1-like viruses during the 2015-2016 season, nasal washes/swabs collected from adult subjects in the Rochester, NY area, were used to sequence and isolate the circulating viruses. The HA and NA proteins from viruses circulating during the 2015-2016 season encoded eighteen and fourteen amino acid differences, respectively, when compared to A/California/04/2009, a strain circulating at the origin of the 2009 pandemic. The circulating strains belonged to subclade 6B.1, defined by HA amino acid substitutions S101N, S179N, and I233T. Hemagglutination-inhibiting (HAI) and HA-specific neutralizing serum antibody (Ab) titers from around 50% of pH1N1-like virus-infected subjects and immune ferrets were 2-4 fold lower for the 2015-2016 circulating strains compared to the vaccine strain. In addition, using a luminex-based mPlex HA assay, the binding of human sera from subjects infected with pH1N1-like viruses to the HA proteins from circulating and vaccine strains was not identical, strongly suggesting antigenic differences in the HA protein. Additionally, NA inhibition (NAI) Ab titers in human sera from pH1N1-like virus-infected subjects increased after the infection and there were measurable antigenic differences between the NA protein of circulating strains and the vaccine strain using both ferret and human antisera. Despite having been vaccinated, infected subjects exhibited low HAI Ab titers against the vaccine and circulating strains. This suggests that poor responses to the H1N1 component of the vaccine as well as antigenic differences in the HA and NA proteins of currently circulating pH1N1-like viruses could be contributing to risk of infection even after vaccination.

Influenza A viruses (IAVs) cause seasonal epidemics and occasional pandemics, representing a serious public health concern. It has been described that one mechanism used by some IAV strains to escape the host innate immune responses and modulate virus pathogenicity involves the ability of the PA-X and NS1 proteins to inhibit the host protein synthesis in infected cells. It was reported that for the 2009 pandemic H1N1 IAV (pH1N1) only the PA-X protein had this inhibiting capability, while the NS1 protein did not. In this work, we have evaluated, for the first time, the combined effect of PA-X- and NS1-mediated inhibition of general gene expression on virus pathogenesis, using a temperature-sensitive, live-attenuated 2009 pandemic H1N1 IAV (pH1N1 LAIV). We found that viruses containing PA-X and NS1 proteins that simultaneously have (PA WT + /NS1 MUT + ) or do not have (PA MUT − /NS1 WT − ) the ability to block host gene expression showed reduced pathogenicity in vivo . However, a virus where the ability to inhibit host protein expression was switched between PA-X and NS1 (PA MUT − /NS1 MUT + ) presented pathogenicity similar to that of a virus containing both wild-type proteins (PA WT + /NS1 WT − ). Our findings suggest that inhibition of host protein expression is subject to a strict balance, which can determine the successful progression of IAV infection. Importantly, knowledge obtained from our studies could be used for the development of new and more effective vaccine approaches against IAV. IMPORTANCE Influenza A viruses (IAVs) are one of the most common causes of respiratory infections in humans, resulting in thousands of deaths annually. Furthermore, IAVs can cause unpredictable pandemics of great consequence when viruses not previously circulating in humans are introduced into humans. The defense machinery provided by the host innate immune system limits IAV replication; however, to counteract host antiviral activities, IAVs have developed different inhibition mechanisms, including prevention of host gene expression mediated by the viral PA-X and NS1 proteins. Here, we provide evidence demonstrating that optimal control of host protein synthesis by IAV PA-X and/or NS1 proteins is required for efficient IAV replication in the host. Moreover, we demonstrate the feasibility of genetically controlling the ability of IAV PA-X and NS1 proteins to inhibit host immune responses, providing an approach to develop more effective vaccines to combat disease caused by this important respiratory pathogen.

The induction of antibodies specific for the influenza HA protein stalk domain is being pursued as a universal strategy against influenza virus infections. However, little work has been done looking at natural or induced antigenic variability in this domain and the effects on viral fitness. We analyzed human H1 HA head and stalk domain sequences and found substantial variability in both, although variability was highest in the head region. Furthermore, using human immune sera from pandemic A/California/04/2009 immune subjects and mAbs specific for the stalk domain, viruses were selected in vitro containing mutations in both domains that partially contributed to immune evasion. Recombinant viruses encoding amino acid changes in the HA stalk domain replicated well in vitro, and viruses incorporating two of the stalk mutations retained pathogenicity in vivo. These findings demonstrate that the HA protein stalk domain can undergo limited drift under immune pressure and the viruses can retain fitness and virulence in vivo, findings which are important to consider in the context of vaccination targeting this domain.

Canine influenza viruses (CIVs) are the causative agents of canine influenza, a contagious respiratory disease in dogs, and include the equine-origin H3N8 and the avian-origin H3N2 viruses. Influenza A virus (IAV) nonstructural protein 1 (NS1) is a virulence factor essential for counteracting the innate immune response. Here, we evaluated the ability of H3N8 CIV NS1 to inhibit host innate immune responses. We found that H3N8 CIV NS1 was able to efficiently counteract interferon (IFN) responses but was unable to block general gene expression in human or canine cells. Such ability was restored by a single amino acid substitution in position 186 (K186E) that resulted in NS1 binding to the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), a cellular protein involved in pre-mRNA processing. We also examined the frequency distribution of K186 and E186 among H3N8 CIVs and equine influenza viruses (EIVs), the ancestors of H3N8 CIV, and experimentally determined the impact of amino acid 186 in the ability of different CIV and EIV NS1s to inhibit general gene expression. In all cases, the presence of E186 was responsible for the control of host gene expression. In contrast, the NS1 protein of H3N2 CIV harbors E186 and blocks general gene expression in canine cells. Altogether, our results confirm previous studies on the strain-dependent ability of NS1 to block general gene expression. Moreover, the observed polymorphism on amino acid 186 between H3N8 and H3N2 CIVs might be the result of adaptive changes acquired during long-term circulation of avian-origin IAVs in mammals. IMPORTANCE Canine influenza is a respiratory disease of dogs caused by two CIV subtypes, the H3N8 and H3N2 viruses, of equine and avian origins, respectively. Influenza NS1 is the main viral factor responsible for the control of host innate immune responses, and changes in NS1 can play an important role in host adaptation. Here we assessed the ability of H3N8 CIV NS1 to inhibit host innate immune responses and gene expression. The H3N8 CIV NS1 did not block host gene expression, but this activity was restored by a single amino acid substitution (K186E), which was responsible for NS1 binding to the host factor CPSF30. In contrast, the H3N2 CIV NS1, which contains E186, blocks general gene expression. Our results suggest that the ability to block host gene expression is not required for influenza virus replication in mammals but might be important in the long-term adaptation of avian-origin influenza viruses to mammals.

In 2009, a novel H1N1 influenza virus emerged in humans, causing a global pandemic. It was previously shown that the NS1 protein from this human 2009 pandemic H1N1 (pH1N1) virus was an effective interferon (IFN) antagonist but could not inhibit general host gene expression, unlike other NS1 proteins from seasonal human H1N1 and H3N2 viruses. Here we show that the NS1 protein from currently circulating pH1N1 viruses has evolved to encode 6 amino acid changes (E55K, L90I, I123V, E125D, K131E, and N205S) with respect to the original protein. Notably, these 6 residue changes restore the ability of pH1N1 NS1 to inhibit general host gene expression, mainly by their ability to restore binding to the cellular factor CPSF30. This is the first report describing the ability of the pH1N1 NS1 protein to naturally acquire mutations that restore this function. Importantly, a recombinant pH1N1 virus containing these 6 amino acid changes in the NS1 protein (pH1N1/NSs-6mut) inhibited host IFN and proinflammatory responses to a greater extent than that with the parental virus (pH1N1/NS1-wt), yet virus titers were not significantly increased in cell cultures or in mouse lungs, and the disease was partially attenuated. The pH1N1/NSs-6mut virus grew similarly to pH1N1/NSs-wt in mouse lungs, but infection with pH1N1/NSs-6mut induced lower levels of proinflammatory cytokines, likely due to a general inhibition of gene expression mediated by the mutated NS1 protein. This lower level of inflammation induced by the pH1N1/NSs-6mut virus likely accounts for the attenuated disease phenotype and may represent a host-virus adaptation affecting influenza virus pathogenesis. IMPORTANCE Seasonal influenza A viruses (IAVs) are among the most common causes of respiratory infections in humans. In addition, occasional pandemics are caused when IAVs circulating in other species emerge in the human population. In 2009, a swine-origin H1N1 IAV (pH1N1) was transmitted to humans, infecting people then and up to the present. It was previously shown that the NS1 protein from the 2009 pandemic H1N1 (pH1N1) virus is not able to inhibit general gene expression. However, currently circulating pH1N1 viruses have evolved to encode 6 amino acid changes (E55K, L90I, I123V, E125D, K131E, and N205S) that allow the NS1 protein of contemporary pH1N1 strains to inhibit host gene expression, which correlates with its ability to interact with CPSF30. Infection with a recombinant pH1N1 virus encoding these 6 amino acid changes (pH1N1/NSs-6mut) induced lower levels of proinflammatory cytokines, resulting in viral attenuation in vivo . This might represent an adaptation of pH1N1 virus to humans.

Influenza virus NS1 protein is a nonstructural, multifunctional protein that counteracts host innate immune responses, modulating virus pathogenesis. NS1 protein variability in subjects infected with H3N2 influenza A viruses (IAVs) during the 2010/2011 season was analyzed, and amino acid changes in residues 86, 189, and 194 were found. The consequences of these mutations for the NS1-mediated inhibition of IFN responses and the pathogenesis of the virus were evaluated, showing that NS1 mutations D189N and V194I impaired the ability of the NS1 protein to inhibit general gene expression, most probably because these mutations decreased the binding of NS1 to the cleavage and polyadenylation specificity factor 30 (CPSF30). A recombinant A/Puerto Rico/8/34 (PR8) H1N1 virus encoding the H3N2 NS1-D189N protein was slightly attenuated, whereas the virus encoding the H3N2 NS1-V194I protein was further attenuated in mice. The higher attenuation of this virus could not be explained by differences in the ability of the two NS1 proteins to counteract host innate immune responses, indicating that another factor must be responsible. In fact, we showed that the virus encoding the H3N2 NS1-V194I protein demonstrated a temperature-sensitive (ts) phenotype, providing a most likely explanation for the stronger attenuation observed. As far as we know, this is the first description of a mutation in NS1 residue 194 conferring a ts phenotype. These studies are relevant in order to identify new residues important for NS1 functions and in human influenza virus surveillance to assess mutations affecting the pathogenicity of circulating viruses. IMPORTANCE Influenza viral infections represent a serious public health problem, with influenza virus causing a contagious respiratory disease that is most effectively prevented through vaccination. The multifunctional nonstructural protein 1 (NS1) is the main viral factor counteracting the host antiviral response. Therefore, influenza virus surveillance to identify new mutations in the NS1 protein affecting the pathogenicity of the circulating viruses is highly important. In this work, we evaluated amino acid variability in the NS1 proteins from H3N2 human seasonal viruses and the effect of the mutations on innate immune responses and virus pathogenesis. NS1 mutations D189N and V194I impaired the ability of the NS1 protein to inhibit general gene expression, and recombinant viruses harboring these mutations were attenuated in a mouse model of influenza infection. Interestingly, a virus encoding the H3N2 NS1-V194I protein demonstrated a temperature-sensitive phenotype, further attenuating the virus in vivo .

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiological agent of a respiratory disease that has a 10% mortality rate. We previously showed that SARS-CoV lacking the E gene (SARS-CoV-¿E) is attenuated in several animal model systems. Here, we show that absence of the E protein resulted in reduced expression of proinflammatory cytokines, decreased numbers of neutrophils in lung infiltrates, diminished lung pathology, and increased mouse survival, suggesting that lung inflammation contributed to SARS-CoV virulence. Further, infection with SARS-CoV-¿E resulted in decreased activation of NF-¿B compared to levels for the wild-type virus. Most important, treatment with drugs that inhibited NF-¿B activation led to a reduction in inflammation and lung pathology in both SARS-CoV-infected cultured cells and mice and significantly increased mouse survival after SARS-CoV infection. These data indicated that activation of the NF-¿B signaling pathway represents a major contribution to the inflammation induced after SARS-CoV infection and that NF-¿B inhibitors are promising antivirals in infections caused by SARS-CoV and potentially other pathogenic human coronaviruses., This work was supported by grants from the Ministry of Science and Innovation of Spain (BIO2010-16705), the Seventh Framework Programme (FP7/2007-2013) of the European Commission (EC) under the project EMPERIE (EC grant agreement number 223498), and the U.S. National Institutes of Health (2P01AI060699-06A1 and CRIP-HHSN266200700010C). M.L.D. received a contract from the project EMPERIE (EC grant agreement number 223498), and J.A.R.-N. and C.C.-R. received a contract from Fundación La Caixa.

Infection of conventional mice with a mouse adapted (MA15) severe acute respiratory syndrome (SARS) coronavirus (CoV) reproduces many aspects of human SARS such as pathological changes in lung, viremia, neutrophilia, and lethality. However, established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection are not available. In this work, efficiently transfectable mouse cell lines stably expressing the murine SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) have been generated. These cells yielded high SARS-CoV-MA15 titers and also served as excellent tools for plaque assays. In addition, in these cell lines, SARS-CoV-MA15 induced the expression of proinflammatory cytokines and IFN-beta, mimicking what has been observed in experimental animal models infected with SARS-CoV and SARS patients. These cell lines are valuable tools to perform in vitro studies in a mouse cell system that reflects the species used for in vivo studies of SARS-CoV-MA15 pathogenesis, This work was supported by grants from the Ministry of Science and Innovation of Spain (BIO2010-16705), the European Community's Seventh Framework Programme (FP7/2007–2013) under the project “EMPERIE” EC Grant Agreement number 223498, and U.S. National Institutes of Health (NIH) (2P01AI060699-06A1, W000306844). JAR received a fellowship from the Fundacion La Caixa