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Avian influenza viruses of the H6 subtype are prevalent in wild ducks and likely play an important role in the ecology of influenza viruses through reassortment with other avian influenza viruses. Yet, only 152 Vietnamese H6 virus sequences were available in GISAID (Global Initiative on Sharing All Influenza Data) prior to this study with the most recent sequences being from 2018. Through surveillance in Vietnamese live bird markets from 2018 to 2021, we identified 287 samples containing one or several H6 viruses and other influenza A virus subtypes, demonstrating a high rate of co-infections among birds in Vietnamese live bird markets. For the 132 H6 samples with unique influenza virus sequences, we conducted phylogenetic and genetic analyses. Most of the H6 viruses were similar to each other and closely related to other H6 viruses; however, signs of reassortment with other avian influenza viruses were evident. At the genetic level, the Vietnamese H6 viruses characterized in our study encode a single basic amino acid at the HA cleavage site, consistent with low pathogenicity in poultry. The Vietnamese H6 viruses analyzed here possess an amino acid motif in HA that confers binding to both avian- and human-type receptors on host cells, consistent with their ability to infect mammals. The frequent detection of H6 viruses in Vietnamese live bird markets, the high rate of co-infections of birds with different influenza viruses, and the dual receptor-binding specificity of these viruses warrant their close monitoring for potential infection and spread among mammals. [ABSTRACT FROM AUTHOR]

Evolution of human H3N2 influenza viruses driven by immune selection has narrowed the receptor specificity of the hemagglutinin (HA) to a restricted subset of human-type (Neu5Acα2-6 Gal) glycan receptors that have extended poly-LacNAc (Galβ1-4GlcNAc) repeats. This altered specificity has presented challenges for hemagglutination assays, growth in laboratory hosts, and vaccine production in eggs. To assess the impact of extended glycan receptors on virus binding, infection, and growth, we have engineered N-glycan extended (NExt) cell lines by overexpressing β3-Ν-acetylglucosaminyltransferase 2 in MDCK, SIAT, and hCK cell lines. Of these, SIAT-NExt cells exhibit markedly increased binding of H3 HAs and susceptibility to infection by recent H3N2 virus strains, but without impacting final virus titers. Glycome analysis of these cell lines and allantoic and amniotic egg membranes provide insights into the importance of extended glycan receptors for growth of recent H3N2 viruses and relevance to their production for cell- and egg-based vaccines. Evolution of human H3N2 influenza has limited the specificity of hemagglutinin to a subset of glycan receptors, which brings challenges. By glyco-engineering cell lines, authors show the importance of extended glycan receptors for growth of recent H3N2 viruses and relevance to their production for vaccines. [ABSTRACT FROM AUTHOR]

H7N9 avian influenza viruses (AIVs) have caused over 1,500 documented human infections since emerging in 2013. Although wild-type H7N9 AIVs can be transmitted by respiratory droplets in ferrets, they have not yet caused widespread outbreaks in humans. Previous studies have revealed molecular determinants of H7N9 AIV host switching, but little is known about potential evolutionary constraints on this process. Here, we compare patterns of sequence evolution for H7N9 AIV and mammalian H1N1 viruses during replication and transmission in ferrets. We show that three main factors—purifying selection, stochasticity, and very narrow transmission bottlenecks—combine to severely constrain the ability of H7N9 AIV to effectively adapt to mammalian hosts in isolated, acute spillover events. We find rare evidence of natural selection favoring new, potentially mammal-adapting mutations within ferrets but no evidence of natural selection acting during transmission. We conclude that human-adapted H7N9 viruses are unlikely to emerge during typical spillover infections. Our findings are instead consistent with a model in which the emergence of a human-transmissible virus would be a rare and unpredictable, though highly consequential, 'jackpot' event. Strategies to control the total number of spillover infections will limit opportunities for the virus to win this evolutionary lottery. [ABSTRACT FROM AUTHOR]

Low pathogenic H9N2 avian influenza viruses have spread in wild birds and poultry worldwide. Recently, the number of human cases of H9N2 virus infection has increased in China and other countries, heightening pandemic concerns. In Japan, H9N2 viruses are not yet enzootic; however, avian influenza viruses, including H5N1, H7N9, H5N6, and H9N2, have been repeatedly detected in raw poultry meat carried by international flight passengers from Asian countries to Japan. Although H9N2 virus-contaminated poultry products intercepted by the animal quarantine service at the Japan border have been characterized in chickens and ducks, the biological properties of those H9N2 viruses in mammals remain unclear. Here, we characterized the biological features of two H9N2 virus isolates [A/chicken/Japan/AQ-HE28-50/2016 (Ck/HE28-50) and A/chicken/Japan/AQ-HE28-57/2016 (Ck/HE28-57)] in a mouse model. We found that these H9N2 viruses replicate well in the respiratory tract of infected mice without adaptation, and that Ck/HE28-57 caused body weight loss in the infected mice. Our results indicate that H9N2 avian influenza viruses isolated from raw chicken meat products illegally brought to Japan can potentially infect and cause disease in mammals. [ABSTRACT FROM AUTHOR]

Exosomes, the extracellular vesicles that contain functional proteins and RNAs, regulate cell-cell communication. Recently, our group reported that levels of various microRNAs (miRNAs) in bronchoalveolar lavage fluid exosomes were highly increased in influenza virus-infected mice and that one of those miRNAs, miR-483-3p, was involved in the potentiation of the innate immune responses to influenza virus infection in mouse type II pneumocytes. Here, we evaluated exosomal miR-483-3p levels in the serum of influenza virus-infected mice and found that miR-483-3p levels were significantly increased during infection with a highly pathogenic avian H5N1 influenza virus. Moreover, miR-483-3p-enriched exosomes derived from type II pneumocytes potentiated the expression of proinflammatory cytokine genes in vascular endothelial cells. Our findings suggest that serum exosomal transfer of miR-483-3p might be involved in the inflammatory pathogenesis of H5N1 influenza virus infection. [ABSTRACT FROM AUTHOR]

Routine surveillance and surveillance in response to influenza outbreaks in avian species in Vietnam in 2009–2013 resulted in the isolation of numerous H5N1 influenza viruses of clades 1.1.2, 2.3.2.1a, 2.3.2.1b, 2.3.2.1c, and 2.3.4.1. Consistent with other studies, we found that viruses of clade 2.3.2.1c were dominant in Vietnam in 2013 and circulated in the northern, central, and southern parts of the country. Phylogenetic analysis revealed reassortment among viruses of clades 2.3.2.1a, 2.3.2.1b, and 2.3.2.1c; in contrast, no reassortment was detected between clade 2.3.2.1 viruses and viruses of clades 1.1.2 or 2.3.4.1, respectively. Deep-sequencing of 42 of the 53 isolated H5N1 viruses revealed viral subpopulations encoding variants that may affect virulence, host range, or sensitivity to antiviral compounds; virus isolates containing these subpopulations may have a higher potential to transmit and adapt to mammals. Among the viruses sequenced, a relatively high number of non-synonymous nucleotide polymorphisms was detected in a virus isolated from a barn swallow, possibly suggesting influenza virus adaption to this host. [ABSTRACT FROM AUTHOR]

H9N2 subtype influenza viruses have been detected in different species of wild birds and domestic poultry in many countries for several decades. Because these viruses are of low pathogenicity in poultry, their eradication is not a priority for animal disease control in many countries, which has allowed them to continue to evolve and spread. Here, we characterized the genetic variation, receptor-binding specificity, replication capability, and transmission in mammals of a series of H9N2 influenza viruses that were detected in live poultry markets in southern China between 2009 and 2013. Thirty-five viruses represented 17 genotypes on the basis of genomic diversity, and one specific “internal-gene-combination” predominated among the H9N2 viruses. This gene combination was also present in the H7N9 and H10N8 viruses that have infected humans in China. All of the 35 viruses preferentially bound to the human-like receptor, although two also retained the ability to bind to the avian-like receptor. Six of nine viruses tested were transmissible in ferrets by respiratory droplet; two were highly transmissible. Some H9N2 viruses readily acquired the 627K or 701N mutation in their PB2 gene upon infection of ferrets, further enhancing their virulence and transmission in mammals. Our study indicates that the widespread dissemination of H9N2 viruses poses a threat to human health not only because of the potential of these viruses to cause an influenza pandemic, but also because they can function as “vehicles” to deliver different subtypes of influenza viruses from avian species to humans. [ABSTRACT FROM AUTHOR]

Cell-surface-receptor binding by influenza viruses is a key determinant of their transmissibility, both from avian and animal species to humans as well as from human to human. Highly pathogenic avian H5N1 viruses that are a threat to public health have been observed to acquire affinity for human receptors, and transmissible-mutant-selection experiments have identified a virus that is transmissible in ferrets, the generally accepted experimental model for influenza in humans. Here, our quantitative biophysical measurements of the receptor-binding properties of haemagglutinin (HA) from the transmissible mutant indicate a small increase in affinity for human receptor and a marked decrease in affinity for avian receptor. From analysis of virus and HA binding data we have derived an algorithm that predicts virus avidity from the affinity of individual HA-receptor interactions. It reveals that the transmissible-mutant virus has a 200-fold preference for binding human over avian receptors. The crystal structure of the transmissible-mutant HA in complex with receptor analogues shows that it has acquired the ability to bind human receptor in the same folded-back conformation as seen for HA from the 1918, 1957 (ref. 4), 1968 (ref. 5) and 2009 (ref. 6) pandemic viruses. This binding mode is substantially different from that by which non-transmissible wild-type H5 virus HA binds human receptor. The structure of the complex also explains how the change in preference from avian to human receptors arises from the Gln226Leu substitution, which facilitates binding to human receptor but restricts binding to avian receptor. Both features probably contribute to the acquisition of transmissibility by this mutant virus. [ABSTRACT FROM AUTHOR]

Pigs have long been considered potential intermediate hosts in which avian influenza viruses can adapt to humans. To determine whether this potential exists for pigs in Indonesia, we conducted surveillance during 2005-2009. We found that 52 pigs in 4 provinces were infected during 2005-2007 but not 2008-2009. Phylogenetic analysis showed that the viruses had been introduced into the pig population in Indonesia on at least 3 occasions. One isolate had acquired the ability to recognize a human-type receptor. No infected pig had influenza-like symptoms, indicating that influenza A (H5N1) viruses can replicate undetected for prolonged periods, facilitating avian virus adaptation to mammalian hosts. Our data suggest that pigs are at risk for infection during outbreaks of influenza virus A (H5N1) and can serve as intermediate hosts in which this avian virus can adapt to mammals. [ABSTRACT FROM AUTHOR]

We investigated whether a vaccine derived from an apathogenic reassortant type A H5N1 influenza strain could induce immune responses in vivo that mediated protection from highly pathogenic avian influenza virus infection in mice. After two subcutaneous immunizations with formalin-inactivated H5N1 whole virus particles (whole particle vaccine), significant killing specific for cells presenting a nucleoprotein peptide from the vaccine strain of the virus was observed. Similar vaccination with viruses treated with ether and formalin, which are commonly used for humans as ether-split vaccines, induced little or no cytotoxic T-cell response. Furthermore, whole particle vaccines of the apathogenic H5N1 strain were more effective than ether-split vaccines at inducing antibody production able to neutralize a highly pathogenic H5N1 strain. Finally, whole particle vaccines of H5N1 protected mice against infection by an H5N1 highly pathogenic avian influenza virus more effectively than did ether-split vaccines. These results suggest that formalin-inactivated virus particles of apathogenic strains are effective for induction of both cytotoxic T-lymphocyte and antibody responses against highly pathogenic avian influenza viruses in vivo, resulting in protection from infection by a highly pathogenic H5N1 virus. [ABSTRACT FROM AUTHOR]

Highly pathogenic avian H5N1 influenza A viruses have spread throughout Asia, Europe, and Africa, raising serious worldwide concern about their pandemic potential. Although more than 250 people have been infected with these viruses, with a consequent high rate of mortality, the molecular mechanisms responsible for the efficient transmission of H5N1 viruses among humans remain elusive. We used a mouse model to examine the role of the amino acid at position 627 of the PB2 viral protein in efficient replication of H5N1 viruses in the mammalian respiratory tract. Viruses possessing Lys at position 627 of PB2 replicated efficiently in lungs and nasal turbinates, as well as in cells, even at the lower temperature of 33 °C. Those viruses possessing Glu at this position replicated less well in nasal turbinates than in lungs, and less well in cells at the lower temperature. These results suggest that Lys at PB2-627 confers to avian H5N1 viruses the advantage of efficient growth in the upper and lower respiratory tracts of mammals. Therefore, efficient viral growth in the upper respiratory tract may provide a platform for the adaptation of avian H5N1 influenza viruses to humans and for efficient person -to-person virus transmission, in the context of changes in other viral properties including specificity for human (sialic acid α-2,6-galactose containing) receptors. [ABSTRACT FROM AUTHOR]

Correction: Genetics, Receptor Binding Property, and Transmissibility in Mammals of Naturally Isolated H9N2 Avian Influenza Viruses The genotype number of DK/ZJ/C1036/09 virus and the maximum body weight change of mice infected with this virus are missing. Graph: Fig 3 Replication and virulence of H9N2 viruses in mice.Virus titers in organs of mice on day 3 p.i. with 106 EID50 of test virus. [Extracted from the article]

Influenza A viruses cause pandemics at random intervals. Pandemics are caused by viruses that contain a hemagglutinin (HA) surface glycoprotein to which human populations are immunologically naive. Such an HA can be introduced into the human population through reassortment between human and avian virus strains or through the direct transfer of an avian influenza virus to humans. The factors that determine the interspecies transmission and pathogenicity of influenza viruses are still poorly understood; however, the HA protein plays an important role in overcoming the interspecies barrier and in virulence in avian influenza viruses. Recently, the RNA polymerase (PB2) protein has also been recognized as a critical factor in host range restriction, while the nonstructural (NS1) protein affects the initial host immune responses. We summarize current knowledge of viral factors that determine host range restriction and pathogenicity of influenza A viruses. [ABSTRACT FROM AUTHOR]

Viral neuraminidase inhibitors show limited efficacy in mice infected with H7N9 influenza A viruses isolated from humans. Although baloxavir marboxil protected mice from lethal challenge infection with a low pathogenic avian influenza H7N9 virus isolated from a human, its efficacy in mice infected with a recent highly pathogenic version of H7N9 human isolates is unknown. Here, we examined the efficacy of baloxavir marboxil in mice infected with a highly pathogenic human H7N9 virus, A/Guangdong/17SF003/2016. Treatment of infected mice with a single 1.5 mg/kg dose of baloxavir marboxil protected mice from the highly pathogenic human H7N9 virus infection as effectively as oseltamivir treatment at 50 mg/kg twice a day for five days. Daily treatment for five days at 15 or 50 mg/kg of baloxavir marboxil showed superior therapeutic efficacy, largely preventing virus replication in respiratory organs. These results indicate that baloxavir marboxil is a valuable candidate treatment for human patients suffering from highly pathogenic H7N9 virus infection. [ABSTRACT FROM AUTHOR]

Highly pathogenic avian H5 influenza viruses persist among poultry and wild birds throughout the world. They sometimes cause interspecies transmission between avian and mammalian hosts. H5 viruses possessing the HA of subclade 2.3.4.4, 2.3.2.1, 2.2.1, or 7.2 were detected between 2015 and 2018. To understand the neutralizing epitopes of H5-HA, we characterized 15 human monoclonal antibodies (mAbs) against the HA of H5 viruses, which were obtained from volunteers who received the H5N1 vaccine that contains a subclade 2.2.1 or 2.1.3.2 virus as an antigen. Twelve mAbs were specific for the HA of subclade 2.2.1, two mAbs were specific for the HA of subclade 2.1.3.2, and one mAb was specific for the HA of both. Of the 15 mAbs analyzed, nine, which were specific for the HA of subclade 2.2.1, and shared the VH and VL genes, possessed hemagglutination inhibition and neutralizing activities, whereas the others did not. A single amino acid substitution or insertion at positions 144–147 in antigenic site A conferred resistance against these nine mAbs to the subclade 2.2.1 viruses. The amino acids at positions 144–147 are highly conserved among subclade 2.2.1, but differ from those of other subclades. These results show that the neutralizing epitope including amino acids at positions 144–147 is targeted by human antibodies, and plays a role in the antigenic difference between subclade 2.2.1 and other subclades. [ABSTRACT FROM AUTHOR]

The article discusses the research conducted by the researchers of NIAID Centers of Excellence in Influence Research and Surveillance regarding the threat posed by the A(H7N9) viruses. It states that the virus has several motifs which are mammalian in characteristics. It also mentions that the haemagglutinin proteins of the virus have cleave site which is consistent with low-pathogenic phenotype in birds.

Although more than 100 people have been infected by H5N1 influenza A viruses, human-to-human transmission is rare. What are the molecular barriers limiting human-to-human transmission? Here we demonstrate an anatomical difference in the distribution in the human airway of the different binding molecules preferred by the avian and human influenza viruses. The respective molecules are sialic acid linked to galactose by an α-2,3 linkage (SAα2,3Gal) and by an α-2,6 linkage (SAα2,6Gal). Our findings may provide a rational explanation for why H5N1 viruses at present rarely infect and spread between humans although they can replicate efficiently in the lungs. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article on the resumption transmission studies for avian flu in the January 2012 issue.

A letter to the editor is presented which discusses the proposal of a 60-day voluntary pause on any research involving the pathogenic avian influenza H5N1 virus.

The persistence of H5N1 avian influenza viruses in many Asian countries and their ability to cause fatal infections in humans have raised serious concerns about a global flu pandemic. Here we report the isolation of an H5N1 virus from a Vietnamese girl that is resistant to the drug oseltamivir, which is an inhibitor of the viral enzyme neuraminidase and is currently used for protection against and treatment of influenza. Further investigation is necessary to determine the prevalence of oseltamivir-resistant H5N1 viruses among patients treated with this drug. [ABSTRACT FROM AUTHOR]

Amino acids at positions 627 and 701 in the PB2 protein (PB2-627 and PB2-701, respectively) of avian influenza A viruses affect virus replication in some mammalian cells. Highly pathogenic H5N1 influenza viruses possessing mammalian-type PB2-627 were detected during the Qinghai Lake outbreak in 2005 and spread to Europe and Africa. Via a database search, we found a high rate of viral isolates from Ratitae, including ostrich, possessing mammalian-type PB2-627 or -701. Here, we report that H5N1 avian influenza viruses possessing mammalian-type amino acids in PB2-627 or -701 are selected during replication in ostrich cells in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Abstract: The wide distribution of H5N1 highly pathogenic avian influenza viruses is a global threat to human health. Indonesia has had the largest number of human infections and fatalities caused by these viruses. To understand the enzootic conditions of the viruses in Indonesia, twenty-four H5N1 viruses isolated from poultry from 2003 to 2007 were phylogenetically characterized. Although previous studies exclusively classified the Indonesian viruses into clades 2.1.1–2.1.3, our phylogenetic analyses showed a new sublineage that did not belong to any of the present clades. In addition, novel reassortant viruses were identified that emerged between this new sublineage and other clades in 2005–2006 on Java Island. H5N1 viruses were introduced from Java Island to Sulawesi, Kalimantan, and Sumatra Island on multiple occasions from 2003–2007, causing the geographical expansion of these viruses in Indonesia. These findings identify Java Island as the epicenter of the Indonesian H5N1 virus expansion. [Copyright &y& Elsevier]

Abstract: A/duck/Guangxi/53/2002 (DKGX/53) and A/duck/Fujian/01/2002 (DKFJ/01) are H5N1 avian influenza viruses that are lethal in chickens. In mice, however, DKFJ/01 is highly pathogenic, whereas DKGX/53 displays low pathogenicity. In this study, we used reverse genetics to demonstrate that two amino acid residues at positions 30 and 215 of the M1 protein of these two viruses are important determinants for pathogenicity in mice. We thus firstly prove the M1 protein contributes to the virulence of H5N1 viruses in mice, and the amino acid residues shown to attenuate the virulence could be targeted in influenza virus candidates for live vaccine development. [Copyright &y& Elsevier]

Abstract: An experimental infection of mice was performed in order to investigate the potential for interspecies transmission in mammals of Italian HPAI viruses of the H7N1 subtype. Three avian origin isolates were selected, two strains obtained from ostrich (one of which contained a PB2-627 Lysine residue) and one from a chicken. Following intranasal infection of mice, clinical signs and mortality were recorded in the experimental groups challenged with the two ostrich isolates, while only weight loss was observed in those receiving the chicken strain. Viruses were recovered to a varying extent from respiratory and nervous tissues of infected animals. These results suggest that HPAI viruses, other than H5N1 and H7N7, may have zoonotic implications, and support the consensus that AI infections in poultry are to be eradicated rather than contained. [Copyright &y& Elsevier]

In 2003, H5N1 avian influenza virus infections were diagnosed in two Hong Kong residents who had visited the Fujian province in mainland China, affording us the opportunity to characterize one of the viral isolates, A/Hong Kong/213/03 (HK213; H5N1). In contrast to H5N1 viruses isolated from humans during the 1997 outbreak in Hong Kong, HK213 retained several features of aquatic bird viruses, including the lack of a deletion in the neuraminidase stalk and the absence of additional oligosaccharide chains at the globular head of the hemagglutinin molecule. It demonstrated weak pathogenicity in mice and ferrets but caused lethal infection in chickens. The original isolate failed to produce disease in ducks but became more pathogenic after five passages. Taken together, these findings portray the HK213 isolate as an aquatic avian influenza A virus without the molecular changes associated with the replication of H5N1 avian viruses in land-based poultry such as chickens. This case challenges the view that adaptation to land-based poultry is a prerequisite for the replication of aquatic avian influenza A viruses in humans. [ABSTRACT FROM AUTHOR]

Viral pneumonia has been frequently reported during early stages of influenza virus pandemics and in many human cases of highly pathogenic avian influenza (HPAI) H5N1 virus infection. To better understand the pathogenesis of this disease, we produced nonlethal viral pneumonia in rhesus macaques by using an HPAI H5N1 virus (A/Anhui/2/2005; referred to as Anhui/2). Infected macaques were monitored for 14 days, and tissue samples were collected at 6 time points for virologic, histopathologic, and transcriptomic analyses. Anhui/2 efficiently replicated in the lung from 12 h to 3 days postinfection (p.i.) and caused temporal but severe pneumonia that began to resolve by day 14. Lung transcriptional changes were first observed at 6 h, and increased expression of vascular permeability regulators and neutrophil chemoattractants correlated with increased serum leakage and neutrophil infiltration in situ. Additional inflammatory, antiviral, and apoptotic genes were upregulated from 12 h, concurrent with viral antigen detection and increasing immune cell populations. A shift toward upregulation of acquired immunity was apparent after day 6. Expression levels of established immune cell molecular markers revealed remarkable similarity with pathological findings, indicating early and robust neutrophil infiltration, a slight delay in macrophage accumulation, and abundant late populations of T lymphocytes. We also characterized the putative mechanisms regulating a unique, pneumonia-associated biphasic fever pattern. Thus, this study is the first to use a comprehensive and integrative approach to delineate specific molecular mechanisms regulating influenza virus-induced pneumonia in nonhuman primates, an important first step toward better management of human influenza virus disease. [ABSTRACT FROM AUTHOR]

Outbreaks of highly pathogenic H5N1 influenza viruses in avian species began in Asia and have since spread to other continents. Concern regarding the pandemic potential of these viruses in humans is clearly warranted, and there is an urgent need to develop effective vaccines against them. Previously, we and others demonstrated that deletions of the M2 cytoplasmic tail caused a growth defect in A/WSN/33 (H1N1) influenza A virus in vitro (K. Iwatsuki-Horimoto, T. Horimoto, T. Noda, M. Kiso, J. Maeda, S. Watanabe, Y. Muramoto, K. Fujii, and Y. Kawaoka, J. Virol. 80:5233-5240, 2006; M. F. McCown and A. Pekosz, J. Virol. 79:3595-3605, 2005; M. F. McCown and A. Pekosz, J. Virol. 80:8178-8189, 2006). We therefore tested the feasibility of using M2 tail mutants as live attenuated vaccines against H5N1 virus. First we generated a series of highly pathogenic H5N1 (A/Vietnam/1203/04 [VN1203]) M2 cytoplasmic tail deletion mutants and examined their growth properties in vitro and in vivo. We found that one mutant, which contains an 11-amino-acid deletion from the C terminus (M2del11 virus), grew as well as the wild-type virus but replicated in mice less efficiently. We then generated a recombinant VN1203M2del11 virus whose hemagglutinin (HA) gene was modified by replacing sequences at the cleavage site with those of an avirulent type of HA (M2del11-HAavir virus). This M2del11-HAavir virus protected mice against challenge with lethal doses of homologous (VN1203; clade 1) and antigenically distinct heterologous (A/Indonesia/7/2005; clade 2) H5N1 viruses. Our results suggest that M2 cytoplasmic tail mutants have potential as live attenuated vaccines against H5N1 influenza viruses. [ABSTRACT FROM AUTHOR]

In this study, we explored the molecular basis determining the virulence of H5N1 avian influenza viruses in mammalian hosts by comparing two viruses, A/Duck/Guangxi/12/03 (DK/12) and A/Duck/Guangxi/27/03 (DK/27), which are genetically similar but differ in their pathogenicities in mice. To assess the genetic basis for this difference in virulence, we used reverse genetics to generate a series of reassortants and mutants of these two viruses. We found that a single-amino-acid substitution of serine for proline at position 42 (P42S) in the NS1 protein dramatically increased the virulence of the DK/12 virus in mice, whereas the substitution of proline for serine at the same position (S42P) completely attenuated the DK/27 virus. We further demonstrated that the amino acid S42 of NS1 is critical for the H5N1 influenza virus to antagonize host cell interferon induction and for the NS1 protein to prevent the double-stranded RNA-mediated activation of the NF-κB pathway and the IRF-3 pathway. Our results indicate that the NS1 protein is critical for the pathogenicity of H5N1 influenza viruses in mammalian hosts and that the amino acid S42 of NS1 plays a key role in undermining the antiviral immune response of the host cell. [ABSTRACT FROM AUTHOR]