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Antigenic drift forces us to frequently update influenza vaccines; however, the genetic basis for antigenic variation remains largely unknown. In this study, we used clade 7.2 H5 viruses as models to explore the molecular determinants of influenza virus antigenic variation. We generated eight monoclonal antibodies (MAbs) targeted to the hemagglutinin (HA) protein of the index virus A/chicken/Shanxi/2/2006 and found that two representative antigenically drifted clade 7.2 viruses did not react with six of the eight MAbs. The E131N mutation and insertion of leucine at position 134 in the HA protein of the antigenically drifted strains eliminated the reactivity of the virus with the MAbs. We also found that the amino acid N131 in the H5 HA protein is glycosylated. Our results provide experimental evidence that glycosylation and an amino acid insertion or deletion in HA influence antigenic variation.

Two amino acids (lysine at position 627 or asparagine at position 701) in the polymerase subunit PB2 protein are considered critical for the adaptation of avian influenza A viruses to mammals. However, the recently emerged pandemic H1N1 viruses lack these amino acids. Here, we report that a basic amino acid at position 591 of PB2 can compensate for the lack of lysine at position 627 and confers efficient viral replication to pandemic H1N1 viruses in mammals. Moreover, a basic amino acid at position 591 of PB2 substantially increased the lethality of an avian H5N1 virus in mice. We also present the X-ray crystallographic structure of the C-terminus of a pandemic H1N1 virus PB2 protein. Arginine at position 591 fills the cleft found in H5N1 PB2 proteins in this area, resulting in differences in surface shape and charge for H1N1 PB2 proteins. These differences may affect the protein's interaction with viral and/or cellular factors, and hence its ability to support virus replication in mammals.

Recent large-scale sequence analyses revealed 'signature' amino acids at specific positions in viral proteins that distinguish human influenza viruses from avian viruses. To determine the role of these host lineage-specific amino acids in the pathogenicity of H5N1 avian influenza viruses, we generated mutant viruses possessing signature amino acids in the PB2, PA and NP proteins of human influenza isolates ('human-like amino acids') in the genetic background of an avian H5N1 virus, and tested their pathogenicity in mice. We found that some of these mutants exhibited enhanced pathogenicity in mice, suggesting the involvement of these host lineage-specific amino acids in the pathogenicity of H5N1 avian influenza viruses in mammals.

The nucleoprotein (NP), which has multiple functions during the virus life cycle, possesses regions that are highly conserved among influenza A, B, and C viruses. To better understand the roles of highly conserved NP amino acids in viral replication, we conducted a comprehensive mutational analysis. Using reverse genetics, we attempted to generate 74 viruses possessing mutations at conserved amino acids of NP. Of these, 48 mutant viruses were successfully rescued; 26 mutants were not viable, suggesting a critical role of the respective NP amino acids in viral replication. To identify the step(s) in the viral life cycle that is impaired by these NP mutations, we examined viral-genome replication/transcription, NP localization, and incorporation of viral-RNA segments into progeny virions. We identified 15 amino acid substitutions in NP that inhibited viral-genome replication and/or transcription, resulting in significant growth defects of viruses possessing these substitutions. We also found several NP mutations that affected the efficient incorporation of multiple viral-RNA (vRNA) segments into progeny virions even though a single vRNA segment was incorporated efficiently. The respective conserved amino acids in NP may thus be critical for the assembly and/or incorporation of sets of eight vRNA segments.

Ebola virus causes lethal hemorrhagic fever in humans, but currently there are no effective vaccines or antiviral compounds for this infectious disease. Passive transfer of monoclonal antibodies (MAbs) protects mice from lethal Ebola virus infection (J. A. Wilson, M. Hevey, R. Bakken, S. Guest, M. Bray, A. L. Schmaljohn, and M. K. Hart, Science 287:1664-1666, 2000). However, the epitopes responsible for neutralization have been only partially characterized because some of the MAbs do not recognize the short synthetic peptides used for epitope mapping. To identify the amino acids recognized by neutralizing and protective antibodies, we generated a recombinant vesicular stomatitis virus (VSV) containing the Ebola virus glycoprotein-encoding gene instead of the VSV G protein-encoding gene and used it to select escape variants by growing it in the presence of a MAb (133/3.16 or 226/8.1) that neutralizes the infectivity of the virus. All three variants selected by MAb 133/3.16 contained a single amino acid substitution at amino acid position 549 in the GP2 subunit. By contrast, MAb 226/8.1 selected three different variants containing substitutions at positions 134, 194, and 199 in the GP1 subunit, suggesting that this antibody recognized a conformational epitope. Passive transfer of each of these MAbs completely protected mice from a lethal Ebola virus infection. These data indicate that neutralizing antibody cocktails for passive prophylaxis and therapy of Ebola hemorrhagic fever can reduce the possibility of the emergence of antigenic variants in infected individuals.

We isolated 77 highly pathogenic avian influenza viruses during routine surveillance in live poultry markets in northern provinces of Vietnam from 2018 to 2021. These viruses are of the H5N6 subtype and belong to HA clades 2.3.4.4g and 2.3.4.4h. Interestingly, we did not detect viruses of clade 2.3.4.4b, which in recent years have dominated in different parts of the world. The viruses isolated in this current study do not encode major determinants of mammalian adaptation (e.g., PB2-E627K or PB1-D701N) but possess amino acid substitutions that may affect viral receptor-binding, replication, or the responses to human antiviral factors. Several of the highly pathogenic H5N6 virus samples contained other influenza viruses, providing an opportunity for reassortment. Collectively, our study demonstrates that the highly pathogenic H5 viruses circulating in Vietnam in 2018–2021 were different from those in other parts of the world, and that the Vietnamese H5 viruses continue to evolve through mutations and reassortment. [ABSTRACT FROM AUTHOR]

The influenza A(H1N1)pdm09 virus that emerged in 2009 causes seasonal epidemic worldwide. The virus acquired several amino acid substitutions that were responsible for antigenic drift until the 2018–2019 influenza season. Viruses possessing mutations in the NA and PA proteins that cause reduced susceptibility to NA inhibitors and baloxavir marboxil, respectively, have been detected after antiviral treatment, albeit infrequently. Here, we analyzed HA, NA, and PA sequences derived from A(H1N1)pdm09 viruses that were isolated during the 2018–2019 and 2019–2020 influenza seasons in Japan. We found that A(H1N1)pdm09 viruses possessing the D187A and Q189E substitutions in HA emerged and dominated during the 2019–2020 season; these substitutions in the antigenic site Sb, a high potency neutralizing antibody-eliciting site for humans, changed the antigenicity of A(H1N1)pdm09 viruses. Furthermore, we found that isolates possessing the N156K substitution, which was predicted to affect the antigenicity of A(H1N1)pdm09 virus at the laboratory level, were detected at a frequency of 1.0% in the 2018–2019 season but 10.1% in the 2019–2020 season. These findings indicate that two kinds of antigenically drifted viruses—N156K and D187A/Q189E viruses—co-circulated during the 2019–2020 influenza season in Japan. [ABSTRACT FROM AUTHOR]

Equine influenza virus is an important pathogen for the horse industry because of its economic impact, and vaccination is a key control measure. Our previous work suggested that a mutation at position 144 in the hemagglutinin of Florida sublineage clade 2 viruses reduces the cross-neutralizing activity of antiserum against a former vaccine strain. To confirm this suggestion, here, we generated viruses by reverse genetics. Antibody titers against the mutated viruses were one-tenth to one-sixteenth of those against the former vaccine strain. Our findings confirm that this single amino acid substitution reduces the cross-reactivity of antiserum against this former Japanese vaccine. [ABSTRACT FROM AUTHOR]

The influenza virus protein PA-X modulates the host immune responses and viral pathogenicity through suppression of host protein expression. The endonuclease active site in the N-terminal region, the basic amino acid cluster in the C-terminal PA-X-specific region, and N-terminal acetylation of PA-X by NatB are important for the shutoff activity of PA-X. Here, we focused on the shutoff activity of PA-X derived from the A/California/04/2009 and A/WSN/33 viruses because these two PA-X proteins differ in their shutoff activity. Mutagenesis analysis revealed that proline and serine at positions 28 and 65, respectively, play a central role in this difference. Furthermore, we found that P28 and S65 also affect the shutoff activity of PA-X derived from other influenza virus subtypes. These data demonstrate that P28 and S65 contribute to enhanced shutoff activity of PA-X. [ABSTRACT FROM AUTHOR]

To clarify the threat posed by emergence of highly pathogenic influenza A(H7N9) virus infection among humans, we characterized the viral polymerase complex. Polymerase basic 2-482R, polymerase basic 2-588V, and polymerase acidic-497R individually or additively enhanced virus polymerase activity, indicating that multiple replication-enhancing mutations in 1 isolate may contribute to virulence. [ABSTRACT FROM AUTHOR]

Abstract: Avian influenza A viruses (IAVs) and human 1918, 1957, and 1968 pandemic IAVs all have neuraminidases (NAs) that are stable at low pH sialidase activity, yet most human epidemic IAVs do not. We examined the pH stability of H5N1 highly pathogenic avian IAV (HPAI) NAs and identified amino acids responsible for conferring stability at low pH. We found that, unlike other avian viruses, most H5N1 IAVs isolated since 2003 had NAs that were unstable at low pH, similar to human epidemic IAVs. These H5N1 viruses are thus already human virus-like and, therefore, have the frequent infections of humans. [Copyright &y& Elsevier]

The influenza virus RNA polymerase complex is a heterotrimer composed of the PB1, PB2, and PA subunits. PB1, the catalytic core and structural backbone of the polymerase, possesses four highly conserved amino acid motifs that are present among all viral RNA-dependent RNA polymerases. A previous study demonstrated the importance of several of these conserved amino acids in PB1 for influenza polymerase activity through mutational analysis. However, a small number of viruses isolated in nature possesses non-consensus amino acids in one of the four motifs, most of which have not been tested for their replicative ability. Here, we assessed the transcription/replication activities of 25 selected PB1 mutations found in natural isolates by using minireplicon assays in human and avian cells. Most of the mutations tested significantly reduced polymerase activity. One exception was mutation K480R, observed in several pandemic (H1N1) 2009 viruses, which slightly increased polymerase activity relative to wild-type. However, in the background of the pandemic A/California/04/2009 (H1N1) virus, this mutation did not affect virus titers in cell culture. Our results further demonstrate the functional importance of the four conserved PB1 motifs in influenza virus transcription/replication. The finding of natural isolates with non-consensus PB1 motifs that are nonfunctional in minireplicon assays suggests compensatory mutations and/or mixed infections which may have 'rescued' the inactive PB1 protein. [ABSTRACT FROM AUTHOR]

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]

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]

We tested the biological significance of two amino acid mutations in the PB2 protein (glutamic acid to lysine at position 627 and aspartic acid to asparagine at position 701) of A(H7N9) viruses for mammalian adaptation. Mutants were assessed for their viral polymerase activities, growth kinetics in mammalian and avian cells, and pathogenicity in mice. We found that lysine at position 627 and asparagine at position 701 in PB2 are essential for mammalian adaptation of A(H7N9) viruses. [ABSTRACT FROM AUTHOR]

The influenza A virus protein PA-X comprises an N-terminal PA region and a C-terminal PA-X-specific region. PA-X suppresses host gene expression, termed shutoff, via mRNA cleavage. Although the endonuclease active site in the N-terminal PA region of PA-X and basic amino acids in the C-terminal PA-X-specific region are known to be important for PA-X shutoff activity, other amino acids may also play a role. Here, we used yeast to identify novel amino acids of PA-X that are important for PA-X shutoff activity. Unlike wild-type PA-X, most PA-X mutants predominantly localized in the cytoplasm, indicating that these mutations decreased the shutoff activity of PA-X by affecting PA-X translocation to the nucleus. Mapping of the identified amino acids onto the N-terminal structure of PA revealed that some of them likely contribute to the formation of the endonuclease active site of PA. [ABSTRACT FROM AUTHOR]

Avian influenza viruses rarely infect humans, but the recently emerged avian H7N9 influenza viruses have caused sporadic infections in humans in China, resulting in 440 confirmed cases with 122 fatalities as of 16 May 2014. In addition, epidemiologic surveys suggest that there have been asymptomatic or mild human infections with H7N9 viruses. These viruses replicate efficiently in mammals, show limited transmissibility in ferrets and guinea pigs, and possess mammalian-adapting amino acid changes that likely contribute to their ability to infect mammals. In this review, we summarize the characteristic features of the novel H7N9 viruses and assess their pandemic potential. [ABSTRACT FROM AUTHOR]

Oseltamivir-resistant H1N1 influenza viruses emerged in 2007 to 2008 and have subsequently circulated widely. However, prior to 2007 to 2008, viruses possessing the neuraminidase (NA) H274Y mutation, which confers oseltamivir resistance, generally had low growth capability. NA mutations that compensate for the deleterious effect of the NA H274Y mutation have since been identified. Given the importance of the functional balance between hemagglutinin (HA) and NA, we focused on amino acid changes in HA. Reverse genetic analysis showed that a mutation at residue 82, 141, or 189 of the HA protein promotes virus replication in the presence of the NA H274Y mutation. Our findings thus identify HA mutations that contributed to the replacement of the oseltamivir-sensitive viruses of 2007 to 2008. [ABSTRACT FROM AUTHOR]

Abstract: In 2009, a swine-origin H1N1 influenza virus caused the first pandemic of the 21st century. To understand the molecular basis of pandemic influenza virus adaptation to new host species, we serially passaged the pandemic (H1N1) 2009 virus strain A/California/04/09 in mouse lungs. After ten passages, the virus became lethal to mice. We found eight amino acid differences between the wild-type and mouse-adapted viruses: one in PB1, three in PA, three in HA, and one in NP. By using reverse genetics to generate mutant viruses, we determined that the amino acid substitutions in PA (at positions 21 and 616), HA (at positions 127 and 222), and NP (at position 375) play independent roles in the increased pathogenicity in mice. Among these five substitutions, an aspartic acid-to-glutamic acid substitution at position 127 in HA contributed to efficient viral replication in mouse lungs. Our results suggest the importance of the viral polymerase complex and of HA in viral adaption to a new host. [Copyright &y& Elsevier]

At 739 amino acids, the nucleoprotein (NP) of Ebola virus is the largest nucleoprotein of the nonsegmented negative-stranded RNA viruses, and like the NPs of other viruses, it plays a central role in virus replication. Huang et al. (Y. Huang, L. Xu, Y. Sun, and G. J. Nabel, Mol. Cell 10:307-316, 2002) previously demonstrated that NP, together with the minor matrix protein VP24 and polymerase cofactor VP35, is necessary and sufficient for the formation of nucleocapsid-like structures that are morphologically indistinguishable from those seen in Ebola virus-infected cells. They further showed that NP is O glycosylated and sialylated and that these modifications are important for interaction between NP and VP35. However, little is known about the structure-function relationship of Ebola virus NP. Here, we examined the glycosylation of Ebola virus NP and further investigated its properties by generating deletion mutants to define the region(s) involved in NP-NP interaction (self-assembly), in the formation of nucleocapsid-like structures, and in the replication of the viral genome. We were unable to identify the types of glycosylation and sialylation, although we did confirm that Ebola virus NP was glycosylated. We also determined that the region from amino acids 1 to 450 is important for NP-NP interaction (self-assembly). We further demonstrated that these amino-terminal 450 residues and the following 150 residues are required for the formation of nucleocapsid-like structures and for viral genome replication. These data advance our understanding of the functional region(s) of Ebola virus NP, which in turn should improve our knowledge of the Ebola virus life cycle and its extreme pathogenicity. [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]

The nucleoprotein (NP), which has multiple functions during the virus life cycle, possesses regions that are highly conserved among influenza A, B, and C viruses. To better understand the roles of highly conserved NP amino acids in viral replication, we conducted a comprehensive mutational analysis. Using reverse genetics, we attempted to generate 74 viruses possessing mutations at conserved amino acids of NP. Of these, 48 mutant viruses were successfully rescued; 26 mutants were not viable, suggesting a critical role of the respective NP amino acids in viral replication. To identify the step(s) in the viral life cycle that is impaired by these NP mutations, we examined viral-genome replication/transcription, NP localization, and incorporation of viral-RNA segments into progeny virions. We identified 15 amino acid substitutions in NP that inhibited viral-genome replication and/or transcription, resulting in significant growth defects of viruses possessing these substitutions. We also found several NP mutations that affected the efficient incorporation of multiple viral-RNA (vRNA) segments into progeny virions even though a single vRNA segment was incorporated efficiently. The respective conserved amino acids in NP may thus be critical for the assembly and/or incorporation of sets of eight vRNA segments. [ABSTRACT FROM AUTHOR]

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]

In order to determine the role of neuraminidase (NA) in host range restriction, we studied a reassortant virus that replicated with poor efficiency in ducks. The reassortant virus, in which NA of an avian virus, A/duck/Hong Kong/278/78 (H2N9), was replaced with that from a human virus, A/England/12/62 (H2N2), was rectally inoculated into ducks. The viruses recovered were then orally inoculated into new ducks and a variant that replicated efficiently in the intestinal tract was isolated. Sequence analysis showed that the variant virus NA contained two amino acid substitutions at positions 165 (Val to Ile) and 431 (Gln to Pro). Differences in low pH resistance of the NAs were also assessed to identify differences in the NAs possibly related to the restriction of viral growth in ducks. The NA activity of the parental reassortant virus almost disappeared after low pH treatment (pH 3.0), while that of the variant was conserved under the same conditions. These results indicate that the amino acids, at positions 165 and/or 431 on the NA molecule, correlate with the ability to support viral growth in ducks, contributing to the low pH stability of NA activity. [Copyright &y& Elsevier]

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]