Influence of viral genome properties on polymerase fidelity.

The first step of viral evolution takes place during genome replication via the error-prone viral polymerase. Among the mutants that arise through this process, only a few well-adapted variants will be selected by natural selection, renewing the viral genome population. Viral polymerase-mediated errors are thought to occur stochastically. However, accumulating evidence suggests that viral polymerase-mediated mutations are heterogeneously distributed throughout the viral genome. Here, we review work that supports this concept and provides mechanistic insights into how specific features of the viral genome could modulate viral polymerase-mediated errors. A predisposition to accumulate viral polymerase-mediated errors at specific loci in the viral genome may guide evolution to specific pathways, thus opening new directions of research to better understand viral evolutionary dynamics.

The Activation of the RIG-I/MDA5 Signaling Pathway upon Influenza D Virus Infection Impairs the Pulmonary Proinflammatory Response Triggered by Mycoplasma bovis Superinfection.

Concurrent infections with multiple pathogens are often described in cattle with respiratory illness. However, how the host-pathogen interactions influence the clinical outcome has been only partially explored in this species. Influenza D virus (IDV) was discovered in 2011. Since then, IDV has been detected worldwide in different hosts. A significant association between IDV and bacterial pathogens in sick cattle was shown in epidemiological studies, especially with Mycoplasma bovis. In an experimental challenge, IDV aggravated M. bovis-induced pneumonia. However, the mechanisms through which IDV drives an increased susceptibility to bacterial superinfections remain unknown. Here, we used the organotypic lung model precision-cut lung slices to study the interplay between IDV and M. bovis coinfection. Our results show that a primary IDV infection promotes M. bovis superinfection by increasing the bacterial replication and the ultrastructural damages in lung pneumocytes. In our model, IDV impaired the innate immune response triggered by M. bovis by decreasing the expression of several proinflammatory cytokines and chemokines that are important for immune cell recruitment and the bacterial clearance. Stimulations with agonists of cytosolic helicases and Toll-like receptors (TLRs) revealed that a primary activation of RIG-I/MDA5 desensitizes the TLR2 activation, similar to what was observed with IDV infection. The cross talk between these two pattern recognition receptors leads to a nonadditive response, which alters the TLR2-mediated cascade that controls the bacterial infection. These results highlight innate immune mechanisms that were not described for cattle so far and improve our understanding of the bovine host-microbe interactions and IDV pathogenesis. IMPORTANCE Since the spread of the respiratory influenza D virus (IDV) infection to the cattle population, the question about the impact of this virus on bovine respiratory disease (BRD) remains still unanswered. Animals affected by BRD are often coinfected with multiple pathogens, especially viruses and bacteria. In particular, viruses are suspected to enhance secondary bacterial superinfections. Here, we use an ex vivo model of lung tissue to study the effects of IDV infection on bacterial superinfections. Our results show that IDV increases the susceptibility to the respiratory pathogen Mycoplasma bovis. In particular, IDV seems to activate immune pathways that inhibit the innate immune response against the bacteria. This may allow M. bovis to increase its proliferation and to delay its clearance from lung tissue. These results suggest that IDV could have a negative impact on the respiratory pathology of cattle.

Opposite Outcomes of the Within-Host Competition between High- and Low-Pathogenic H5N8 Avian Influenza Viruses in Chickens Compared to Ducks.

Highly pathogenic avian influenza viruses (HPAIV) emerge from low-pathogenic avian influenza viruses (LPAIV) through the introduction of basic amino acids at the hemagglutinin (HA) cleavage site. Following viral evolution, the newly formed HPAIV likely represents a minority variant within the index host, predominantly infected with the LPAIV precursor. Using reverse genetics-engineered H5N8 viruses differing solely at the HA cleavage, we tested the hypothesis that the interaction between the minority HPAIV and the majority LPAIV could modulate the risk of HPAIV emergence and that the nature of the interaction could depend on the host species. In chickens, we observed that the H5N8LP increased H5N8HP replication and pathogenesis. In contrast, the H5N8LP antagonized H5N8HP replication and pathogenesis in ducks. Ducks mounted a more potent antiviral innate immune response than chickens against the H5N8LP, which correlated with H5N8HP inhibition. These data provide experimental evidence that HPAIV may be more likely to emerge in chickens than in ducks and underscore the importance of within-host viral variant interactions in viral evolution. IMPORTANCE Highly pathogenic avian influenza viruses represent a threat to poultry production systems and to human health because of their impact on food security and because of their zoonotic potential. It is therefore crucial to better understand how these viruses emerge. Using a within-host competition model between high- and low-pathogenic avian influenza viruses, we provide evidence that highly pathogenic avian influenza viruses could be more likely to emerge in chickens than in ducks. These results have important implications for highly pathogenic avian influenza virus emergence prevention, and they underscore the importance of within-host viral variant interactions in virus evolution.

Intranasal type I interferon treatment is beneficial only when administered before clinical signs onset in the SARS-CoV-2 hamster model.

Impaired type I interferons (IFNs) production or signaling have been associated with severe COVID-19, further promoting the evaluation of recombinant type I IFNs as therapeutics against SARS-CoV-2 infection. In the Syrian hamster model, we show that intranasal administration of IFN-α starting one day pre-infection or one day post-infection limited weight loss and decreased viral lung titers. By contrast, intranasal administration of IFN-α starting at the onset of symptoms three days post-infection had no impact on the clinical course of SARS-CoV-2 infection. Our results provide evidence that early type I IFN treatment is beneficial, while late interventions are ineffective, although not associated with signs of enhanced disease.

A New Derivative of Retro-2 Displays Antiviral Activity against Respiratory Syncytial Virus.

Human respiratory syncytial virus (hRSV) is the most common cause of bronchiolitis and pneumonia in newborns, with all children being infected before the age of two. Reinfections are very common throughout life and can cause severe respiratory infections in the elderly and immunocompromised adults. Although vaccines and preventive antibodies have recently been licensed for use in specific subpopulations of patients, there is still no therapeutic treatment commonly available for these infections. Here, we investigated the potential antiviral activity of Retro-2.2, a derivative of the cellular retrograde transport inhibitor Retro-2, against hRSV. We show that Retro-2.2 inhibits hRSV replication in cell culture and impairs the ability of hRSV to form syncytia. Our results suggest that Retro-2.2 treatment affects virus spread by disrupting the trafficking of the viral de novo synthetized F and G glycoproteins to the plasma membrane, leading to a defect in virion morphogenesis. Taken together, our data show that targeting intracellular transport may be an effective strategy against hRSV infection.

Pathobiology of highly pathogenic H5 avian influenza viruses in naturally infected Galliformes and Anseriformes in France during 2015-2016.

In late 2015, an epizootic of Highly Pathogenic Avian Influenza (H5Nx) was registered in Southwestern France, including more than 70 outbreaks in commercial poultry flocks. Phylogenetic analyses suggested local emergence of H5 viruses which differed from A/goose/Guangdong/1/1996 clade 2.3.4.4b lineage and shared a unique polybasic cleavage site in their hemagglutinin protein. The present work provides an overview of the pathobiological picture associated with this epizootic in naturally infected chickens, guinea fowls and ducks. Upon necropsy examination, selected tissues were sampled for histopathology, immunohistochemistry and quantitative Real Time Polymerase Chain Reaction. In Galliformes, HPAIVs infection manifested as severe acute systemic vasculitis and parenchymal necrosis and was associated with endothelial expression of viral antigen. In ducks, lesions were mild and infrequent, with sparse antigenic detection in respiratory and digestive mucosae and leukocytes. Tissue quantifications of viral antigen and RNA were higher in chickens and guinea fowls compared to duck. Subsequently, recombinant HA (rHA) was generated from a H5 HPAIV isolated from an infected duck to investigate its glycan-binding affinity for avian mucosae. Glycan-binding analysis revealed strong affinity of rHA for 3'Sialyl-LacNAc and low affinity for Sialyl-LewisX, consistent with a duck-adapted virus similar to A/Duck/Mongolia/54/2001 (H5N2). K222R and S227R mutations on rHA sequence shifted affinity towards Sialyl-LewisX and led to an increased affinity for chicken mucosa, confirming the involvement of these two mutations in the glycan-binding specificity of the HA. Interestingly, the rHA glycan binding pattern of guinea fowl appeared intermediate between duck and chicken. The present study presents a unique pathobiological description of the H5 HPAIVs outbreaks that occurred in 2015-2016 in Southwestern France.

Controlling the emergence of highly pathogenic avian influenza viruses: one of the challenges of the 21st century? / La lutte contre l'émergence des virus influenza aviaires hautement pathogènes : un des défis du XXIe siècle ?

Millions of farmed birds culled, hundreds of gannets dead on the coast, dying marine mammals suffering from neurological disorders: these events regularly make the headlines. What do they have in common? Highly pathogenic avian influenza viruses (HPAIV). HPAIVs are viruses capable of replicating systemically, causing both asymptomatic infections and devastating mortality, depending on the susceptibility of the host species. Known for several decades now, these viruses have seen their circulation particularly increased in recent years, and have been responsible for massive epizootics on several continents. In addition to the devastating effects they can cause in poultry and wildlife, HPAIVs are also capable of crossing the species barrier. Improving knowledge about these viruses and better control of their spread therefore has several objectives: to protect public health, to guarantee food safety, to preserve biodiversity and the economy of the poultry industry. This article reviews the current state of knowledge on HPAIVs: from their epidemiology to the mechanisms of emergence and control measures.

Des millions d'oiseaux d'élevage abattus, des centaines de fous de Bassan morts sur le littoral, des mammifères marins agonisants atteints de troubles neurologiques : ces événements font régulièrement la une de l'actualité. Leur point commun ? Les virus influenza aviaires hautement pathogènes (VIAHP). Les VIAHP sont des virus capables de se répliquer de manière systémique, engendrant des infections asymptomatiques comme une mortalité foudroyante, en fonction de la sensibilité des espèces hôtes. Connus depuis maintenant plusieurs décennies, ces virus ont vu leur circulation particulièrement augmenter ces dernières années et ont été responsables d'épizooties massives sur plusieurs continents. Aux effets dévastateurs qu'ils peuvent causer dans les filières avicoles et dans la faune sauvage, les VIAHP sont également capables de franchir la barrière d'espèce. Mieux les connaître et mieux maîtriser leur diffusion revêt donc plusieurs objectifs : protéger la santé publique, garantir la sécurité alimentaire, préserver la biodiversité ou encore l'économie des filières avicoles. Cet article de synthèse fait un état de l'art des connaissances actuelles sur les VIAHP : de leur épidémiologie aux mécanismes d'émergence en passant par les mesures de lutte.

The Microbiota Contributes to the Control of Highly Pathogenic H5N9 Influenza Virus Replication in Ducks.

Ducks usually show little or no clinical signs following highly pathogenic avian influenza virus infection. In order to analyze whether the microbiota could contribute to the control of influenza virus replication in ducks, we used a broad-spectrum oral antibiotic treatment to deplete the microbiota before infection with a highly pathogenic H5N9 avian influenza virus. Antibiotic-treated ducks and nontreated control ducks did not show any clinical signs following H5N9 virus infection. We did not detect any significant difference in virus titers neither in the respiratory tract nor in the brain nor spleen. However, we found that antibiotic-treated H5N9 virus-infected ducks had significantly increased intestinal virus excretion at days 3 and 5 postinfection. This was associated with a significantly decreased antiviral immune response in the intestine of antibiotic-treated ducks. Our findings highlight the importance of an intact microbiota for an efficient control of avian influenza virus replication in ducks.IMPORTANCE Ducks are frequently infected with avian influenza viruses belonging to multiple subtypes. They represent an important reservoir species of avian influenza viruses, which can occasionally be transmitted to other bird species or mammals, including humans. Ducks thus have a central role in the epidemiology of influenza virus infection. Importantly, ducks usually show little or no clinical signs even following infection with a highly pathogenic avian influenza virus. We provide evidence that the microbiota contributes to the control of influenza virus replication in ducks by modulating the antiviral immune response. Ducks are able to control influenza virus replication more efficiently when they have an intact intestinal microbiota. Therefore, maintaining a healthy microbiota by limiting perturbations to its composition should contribute to the prevention of avian influenza virus spread from the duck reservoir.

Massive transient damage of the olfactory epithelium associated with infection of sustentacular cells by SARS-CoV-2 in golden Syrian hamsters.

Anosmia is one of the most prevalent symptoms of SARS-CoV-2 infection during the COVID-19 pandemic. However, the cellular mechanism behind the sudden loss of smell has not yet been investigated. The initial step of odour detection takes place in the pseudostratified olfactory epithelium (OE) mainly composed of olfactory sensory neurons surrounded by supporting cells known as sustentacular cells. The olfactory neurons project their axons to the olfactory bulb in the central nervous system offering a potential pathway for pathogens to enter the central nervous system by bypassing the blood brain barrier. In the present study, we explored the impact of SARS-CoV-2 infection on the olfactory system in golden Syrian hamsters. We observed massive damage of the OE as early as 2 days post nasal instillation of SARS-CoV-2, resulting in a major loss of cilia necessary for odour detection. These damages were associated with infection of a large proportion of sustentacular cells but not of olfactory neurons, and we did not detect any presence of the virus in the olfactory bulbs. We observed massive infiltration of immune cells in the OE and lamina propria of infected animals, which may contribute to the desquamation of the OE. The OE was partially restored 14 days post infection. Anosmia observed in COVID-19 patient is therefore likely to be linked to a massive and fast desquamation of the OE following sustentacular cells infection with SARS-CoV-2 and subsequent recruitment of immune cells in the OE and lamina propria.

Membrane lipid saturation activates endoplasmic reticulum unfolded protein response transducers through their transmembrane domains.

Endoplasmic reticulum (ER) stress sensors use a related luminal domain to monitor the unfolded protein load and convey the signal to downstream effectors, signaling an unfolded protein response (UPR) that maintains compartment-specific protein folding homeostasis. Surprisingly, perturbation of cellular lipid composition also activates the UPR, with important consequences in obesity and diabetes. However, it is unclear if direct sensing of the lipid perturbation contributes to UPR activation. We found that mutant mammalian ER stress sensors, IRE1α and PERK, lacking their luminal unfolded protein stress-sensing domain, nonetheless retained responsiveness to increased lipid saturation. Lipid saturation-mediated activation in cells required an ER-spanning transmembrane domain and was positively regulated in vitro by acyl-chain saturation in reconstituted liposomes. These observations suggest that direct sensing of the lipid composition of the ER membrane contributes to the UPR.

Deletion of the C-terminal ESEV domain of NS1 does not affect the replication of a low-pathogenic avian influenza virus H7N1 in ducks and chickens.

Highly pathogenic avian influenza (HPAI) H7N1 viruses caused a series of epizootics in Italy between 1999 and 2001. The emergence of these HPAI viruses coincided with the deletion of the six amino acids R(225)VESEV(230) at the C terminus of NS1. In order to assess how the truncation of NS1 affected virus replication, we used reverse genetics to generate a wild-type low-pathogenic avian influenza (LPAI) H7N1 virus with a 230aa NS1 (H7N1(230)) and a mutant virus with a truncated NS1 (H7N1(224)). The 6aa truncation had no impact on virus replication in duck or chicken cells in vitro. The H7N1(230) and H7N1(224) viruses also replicated to similar levels and induced similar immune responses in ducks or chickens. No significant histological lesions were detected in infected ducks, regardless of the virus inoculated. However, in chickens, the H7N1(230) virus induced a more severe interstitial pneumonia than did the H7N1(224) virus. These findings indicate that the C-terminal extremity of NS1, including the PDZ-binding motif ESEV, is dispensable for efficient replication of an LPAI virus in ducks and chickens, even though it may increase virulence in chickens, as revealed by the intensity of the histological lesions.

In Vitro and In Vivo Characterization of H5N8 High-Pathogenicity Avian Influenza Virus Neurotropism in Ducks and Chickens.

H5N8 high-pathogenicity avian influenza virus (HPAIV) of clade 2.3.4.4B, which circulated during the 2016 epizootics in Europe, was notable for causing different clinical signs in ducks and chickens. The clinical signs preceding death were predominantly neurological in ducks versus respiratory in chickens. To investigate the determinants for the predominant neurological signs observed in ducks, we infected duck and chicken primary cortical neurons. Viral replication was identical in neuronal cultures from both species. In addition, we did not detect any major difference in the immune and inflammatory responses. These results suggest that the predominant neurological involvement of H5N8 HPAIV infection in ducks could not be recapitulated in primary neuronal cultures. In vivo, H5N8 HPAIV replication in ducks peaked soon after infection and led to an early colonization of the central nervous system. In contrast, viral replication was delayed in chickens but ultimately burst in the lungs of chickens, and the chickens died of respiratory distress before brain damage became significant. Consequently, the immune and inflammatory responses in the brain were significantly higher in duck brains than those in chickens. Our study thus suggests that early colonization of the central nervous system associated with prolonged survival after the onset of virus replication is the likely primary cause of the sustained inflammatory response and subsequent neurological disorders observed in H5N8 HPAIV-infected ducks. IMPORTANCE The severity of high-pathogenicity avian influenza virus (HPAIV) infection has been linked to its ability to replicate systemically and cause lesions in a variety of tissues. However, the symptomatology depends on the host species. The H5N8 virus of clade 2.3.4.4B had a pronounced neurotropism in ducks, leading to severe neurological disorders. In contrast, neurological signs were rarely observed in chickens, which suffered mostly from respiratory distress. Here, we investigated the determinants of H5N8 HPAIV neurotropism. We provide evidence that the difference in clinical signs was not due to a difference in neurotropism. Our results rather indicate that chickens died of respiratory distress due to intense viral replication in the lungs before viral replication in the brain could produce significant lesions. In contrast, ducks better controlled virus replication in the lungs, thus allowing the virus to replicate for a sufficient duration in the brain, to reach high levels, and to cause significant lesions.

EPAC1 Pharmacological Inhibition with AM-001 Prevents SARS-CoV-2 and Influenza A Virus Replication in Cells.

The exceptional impact of the COVID-19 pandemic has stimulated an intense search for antiviral molecules. Host-targeted antiviral molecules have the potential of presenting broad-spectrum antiviral activity and are also considered as less likely to select for resistant viruses. In this study, we investigated the antiviral activity exerted by AM-001, a specific pharmacological inhibitor of EPAC1, a host exchange protein directly activated by cyclic AMP (cAMP). The cAMP-sensitive protein, EPAC1 regulates various physiological and pathological processes but its role in SARS-CoV-2 and influenza A virus infection has not yet been studied. Here, we provide evidence that the EPAC1 specific inhibitor AM-001 exerts potent antiviral activity against SARS-CoV-2 in the human lung Calu-3 cell line and the African green monkey Vero cell line. We observed a concentration-dependent inhibition of SARS-CoV-2 infectious viral particles and viral RNA release in the supernatants of AM-001 treated cells that was not associated with a significant impact on cellular viability. Furthermore, we identified AM-001 as an inhibitor of influenza A virus in Calu-3 cells. Altogether these results identify EPAC1 inhibition as a promising therapeutic target against viral infections.

Immune response in the duck intestine following infection with low-pathogenic avian influenza viruses or stimulation with a Toll-like receptor 7 agonist administered orally.

This study analysed the immune response in the intestinal tract of ducks infected with low-pathogenic avian influenza viruses compared with ducks treated orally with R848, a synthetic Toll-like receptor 7 (TLR7) agonist. Influenza virus infection induced a type I interferon (IFN)-dependent immune response characterized by the expression of Mx transcripts in the ileum at levels that were proportional to viral load. Mx transcripts were detected in differentiated enterocytes from influenza virus-infected ducks. By contrast, in R848-treated ducks, Mx transcripts were detected solely in intraepithelial round cells of haematopoietic origin. An increase was detected in the number of intraepithelial TLR7-positive cells and intraepithelial IFN-α-producing cells in influenza virus-infected ducks, albeit to a lower level than in R848-treated ducks. IFN-γ expression was also upregulated in the intestine of influenza virus-infected and R848-treated ducks. Finally, interleukin (IL)-1ß and IL-8 transcripts were expressed at high levels in R848-treated ducks but were not increased in influenza virus-infected ducks. These findings suggest that a type I IFN-mediated immune response in enterocytes and the activation of IFN-γ-secreting cells contribute to the control of influenza virus replication in the duck intestine.

Species-specific contribution of the four C-terminal amino acids of influenza A virus NS1 protein to virulence.

Large-scale sequence analyses of influenza viruses revealed that nonstructural 1 (NS1) proteins from avian influenza viruses have a conserved C-terminal ESEV amino acid motif, while NS1 proteins from typical human influenza viruses have a C-terminal RSKV motif. To test the influence of the C-terminal domains of NS1 on the virulence of an avian influenza virus, we generated a wild-type H7N1 virus with an ESEV motif and a mutant virus with an NS1 protein containing a C-terminal RSKV motif by reverse genetics. We compared the phenotypes of these viruses in vitro in human, mouse, and duck cells as well as in vivo in mice and ducks. In human cells, the human C-terminal RSKV domain increased virus replication. In contrast, the avian C-terminal ESEV motif of NS1 increased virulence in mice. We linked this increase in pathogenicity in mice to an increase in virus replication and to a more severe lung inflammation associated with a higher level of production of type I interferons. Interestingly, the human C-terminal RSKV motif of NS1 increased viral replication in ducks. H7N1 virus with a C-terminal RSKV motif replicated to higher levels in ducks and induced higher levels of Mx, a type I interferon-stimulated gene. Thus, we identify the C-terminal domain of NS1 as a species-specific virulence domain.

Phylodynamic study of the conserved RNA structure encompassing the hemagglutinin cleavage site encoding region of H5 and H7 low pathogenic avian influenza viruses.

Highly pathogenic avian influenza viruses (HPAIVs) evolve from low pathogenic avian influenza viruses (LPAIVs) of the H5 and H7 subtypes. This evolution is characterized by the acquisition of a multi-basic cleavage site (MBCS) motif in the hemagglutinin (HA) that leads to an extended viral tropism and severe disease in poultry. One key unanswered question is whether the risk of transition to HPAIVs is similar for all LPAIVs H5 or H7 strains, or whether specific determinants in the HA sequence of some H5 or H7 LPAIV strains correlate with a higher risk of transition to HPAIVs. Here, we determined if specific features of the conserved RNA stem-loop located at the HA cleavage site-encoding region could be detected along the LPAIV to HPAIV evolutionary pathway. Analysis of the thermodynamic stability of the predicted RNA structures showed no specific patterns common to HA sequences leading to HPAIVs and distinct from those remaining LPAIVs. However, RNA structure clustering analysis revealed that most of the American lineage ancestors leading to H7 emergences via recombination shared the same viral RNA (vRNA) structure topology at the HA1/HA2 boundary region. Our study thus identified predicted secondary RNA structures present in the HA of H7 viruses, which could promote genetic recombination and acquisition of a multibasic cleavage site motif (MBCS).

Immunogenicity and Protective Potential of Mucosal Vaccine Formulations Based on Conserved Epitopes of Influenza A Viruses Fused to an Innovative Ring Nanoplatform in Mice and Chickens.

Current inactivated vaccines against influenza A viruses (IAV) mainly induce immune responses against highly variable epitopes across strains and are mostly delivered parenterally, limiting the development of an effective mucosal immunity. In this study, we evaluated the potential of intranasal formulations incorporating conserved IAV epitopes, namely the long alpha helix (LAH) of the stalk domain of hemagglutinin and three tandem repeats of the ectodomain of the matrix protein 2 (3M2e), as universal mucosal anti-IAV vaccines in mice and chickens. The IAV epitopes were grafted to nanorings, a novel platform technology for mucosal vaccination formed by the nucleoprotein (N) of the respiratory syncytial virus, in fusion or not with the C-terminal end of the P97 protein (P97c), a recently identified Toll-like receptor 5 agonist. Fusion of LAH to nanorings boosted the generation of LAH-specific systemic and local antibody responses as well as cellular immunity in mice, whereas the carrier effect of nanorings was less pronounced towards 3M2e. Mice vaccinated with chimeric nanorings bearing IAV epitopes in fusion with P97c presented modest LAH- or M2e-specific IgG titers in serum and were unable to generate a mucosal humoral response. In contrast, N-3M2e or N-LAH nanorings admixed with Montanide™ gel (MG) triggered strong specific humoral responses, composed of serum type 1/type 2 IgG and mucosal IgG and IgA, as well as cellular responses dominated by type 1/type 17 cytokine profiles. All mice vaccinated with the [N-3M2e + N-LAH + MG] formulation survived an H1N1 challenge and the combination of both N-3M2e and N-LAH nanorings with MG enhanced the clinical and/or virological protective potential of the preparation in comparison to individual nanorings. Chickens vaccinated parenterally or mucosally with N-LAH and N-3M2e nanorings admixed with Montanide™ adjuvants developed a specific systemic humoral response, which nonetheless failed to confer protection against heterosubtypic challenge with a highly pathogenic H5N8 strain. Thus, while the combination of N-LAH and N-3M2e nanorings with Montanide™ adjuvants shows promise as a universal mucosal anti-IAV vaccine in the mouse model, further experiments have to be conducted to extend its efficacy to poultry.

Nucleolar localization of influenza A NS1: striking differences between mammalian and avian cells.

In mammalian cells, nucleolar localization of influenza A NS1 requires the presence of a C-terminal nucleolar localization signal. This nucleolar localization signal is present only in certain strains of influenza A viruses. Therefore, only certain NS1 accumulate in the nucleolus of mammalian cells. In contrast, we show that all NS1 tested in this study accumulated in the nucleolus of avian cells even in the absence of the above described C-terminal nucleolar localization signal. Thus, nucleolar localization of NS1 in avian cells appears to rely on a different nucleolar localization signal that is more conserved among influenza virus strains.

Truncation of the NS1 protein converts a low pathogenic avian influenza virus into a strong interferon inducer in duck cells.

The NS1 protein of influenza A viruses is known as a nonessential virulence factor inhibiting type I interferon (IFN) production in mammals and in chicken cells. Whether NS1 inhibits the induction of type I IFNs in duck cells is currently unknown. In order to investigate this issue, we used reverse genetics to generate a virus expressing a truncated NS1 protein. Using the low pathogenic avian influenza virus A/turkey/Italy/977/1999 (H7N1) as a backbone, we were able to rescue a virus expressing a truncated NS1 protein of 99 amino acids in length. The truncated virus replicated poorly in duck embryonic fibroblasts, but reached high titers in the mammalian IFN-deficient Vero cell line. Using a gene reporter system to measure duck type I IFN production, we showed that the truncated virus is a potent inducer of type I IFN in cell culture. These results show that the NS1 protein functions to prevent the induction of IFN in duck cells and underline the need for a functional NS1 protein in order for the virus to express its full virulence.