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Virulence-affecting amino acid changes in the PA protein of H7N9 influenza A viruses.

ABSTRACT: Novel avian-origin influenza A(H7N9) viruses were first reported to infect humans in March 2013. To date, 143 human cases, including 45 deaths, have been recorded. By using sequence comparisons and phylogenetic and ancestral inference analyses, we identified several distinct amino acids in the A(H7N9) polymerase PA protein, some of which may be mammalian adapting. Mutant viruses possessing some of these amino acid changes, singly or in combination, were assessed for their polymerase activities and growth kinetics in mammalian and avian cells and for their virulence in mice. We identified several mutants that were slightly more virulent in mice than the wild-type A(H7N9) virus, A/Anhui/1/2013. These mutants also exhibited increased polymerase activity in human cells but not in avian cells. Our findings indicate that the PA protein of A(H7N9) viruses has several amino acid substitutions that are attenuating in mammals.Novel avian-origin influenza A(H7N9) viruses emerged in the spring of 2013. By using computational analyses of A(H7N9) viral sequences, we identified several amino acid changes in the polymerase PA protein, which we then assessed for their effects on viral replication in cultured cells and mice. We found that the PA proteins of A(H7N9) viruses possess several amino acid substitutions that cause attenuation in mammals.

SUBMITTER: Yamayoshi S

PROVIDER: S-EPMC3957961 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Virulence-affecting amino acid changes in the PA protein of H7N9 influenza A viruses.

Yamayoshi Seiya S Yamada Shinya S Fukuyama Satoshi S Murakami Shin S Zhao Dongming D Uraki Ryuta R Watanabe Tokiko T Tomita Yuriko Y Macken Catherine C Neumann Gabriele G Kawaoka Yoshihiro Y

Journal of virology 20131226 6

<h4>Unlabelled</h4>Novel avian-origin influenza A(H7N9) viruses were first reported to infect humans in March 2013. To date, 143 human cases, including 45 deaths, have been recorded. By using sequence comparisons and phylogenetic and ancestral inference analyses, we identified several distinct amino acids in the A(H7N9) polymerase PA protein, some of which may be mammalian adapting. Mutant viruses possessing some of these amino acid changes, singly or in combination, were assessed for their poly ...[more]

PMID: 24371069

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Project description: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.

Project description:The potential avian influenza pandemic remains a threat to public health, as the avian-origin influenza A(H7N9) virus has caused more than 1,560 laboratory-confirmed human infections since 2013, with nearly 40% mortality. Development of low-pathogenic candidate vaccine viruses (CVVs) for vaccine production is essential for pandemic preparedness. However, the suboptimal growth of CVVs in mammalian cells and chicken eggs is often a challenge. By introducing a single adaptive substitution, G218E, into the hemagglutinin (HA), we generated reassortant A(H7N9)-G218E CVVs that were characterized by significantly enhanced growth in both cells and eggs. These G218E CVVs retained the original antigenicity, as determined by a hemagglutination inhibition assay, and effectively protected ferrets from lethal challenge with the highly pathogenic parental virus. We found that the suboptimal replication of the parental H7 CVVs was associated with impeded progeny virus release as a result of strong HA receptor binding relative to weak neuraminidase (NA) cleavage of receptors. In contrast, the G218E-mediated growth improvement was attributed to relatively balanced HA and NA functions, resulted from reduced HA binding to both human- and avian-type receptors, and thus facilitated NA-mediated virus release. Our findings revealed that a single amino acid mutation at residue 218 of the HA improved the growth of A(H7N9) influenza virus by balancing HA and NA functions, shedding light on an alternative approach for optimizing certain influenza CVVs.IMPORTANCE The circulating avian influenza A(H7N9) has caused recurrent epidemic waves with high mortality in China since 2013, in which the alarming fifth wave crossing 2016 and 2017 was highlighted by a large number of human infections and the emergence of highly pathogenic avian influenza (HPAI) A(H7N9) strains in human cases. We generated low-pathogenic reassortant CVVs derived from the emerging A(H7N9) with improved virus replication and protein yield in both MDCK cells and eggs by introducing a single substitution, G218E, into HA, which was associated with reducing HA receptor binding and subsequently balancing HA-NA functions. The in vitro and in vivo experiments demonstrated comparable antigenicity of the G218E CVVs with that of their wild-type (WT) counterparts, and both the WT and the G218E CVVs fully protected ferrets from parental HPAI virus challenge. With high yield traits and the anticipated antigenicity, the G218E CVVs should benefit preparedness against the threat of an A(H7N9) influenza pandemic.

Dataset Information

Virulence-affecting amino acid changes in the PA protein of H7N9 influenza A viruses.

Publications

Virulence-affecting amino acid changes in the PA protein of H7N9 influenza A viruses.

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