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Amino Acid Mutations A286V and T437M in the Nucleoprotein Attenuate H7N9 Viruses in Mice.

ABSTRACT: The low-pathogenic H7N9 influenza viruses that emerged in 2013 acquired an insertion of four amino acids in their hemagglutinin cleavage site and thereby became highly pathogenic to chickens in 2017. Previous studies indicated that these highly pathogenic H7N9 viruses are virulent in chickens but have distinct pathotypes in mice. A/chicken/Guangdong/SD098/2017 (CK/SD098) is avirulent, with a 50% mouse lethal dose (MLD₅₀) of >7.5 log₁₀ 50% egg infectious dose (EID₅₀), whereas A/chicken/Hunan/S1220/2017 (CK/S1220) is virulent in mice, with an MLD₅₀ of 3.2 log₁₀ EID₅₀ In this study, we explored the genetic determinants that contribute to the difference in virulence between these two H7N9 viruses by generating a series of reassortants and mutants in the CK/S1220 virus background and testing their virulence in mice. We found that the reassortant CK/1220-SD098-NP, carrying the nucleoprotein (NP) of CK/SD098, was avirulent in mice, with an MLD₅₀ of >10^7.5 EID₅₀ The NPs of these two viruses differ by two amino acids, at positions 286 and 437. We further demonstrated that the amino acid mutations A286V and T437M of NP independently slowed the process of NP import to and export from the nucleus and thus jointly impaired the viral life cycle and attenuated the virulence of these H7N9 viruses in mice. Our study identified new virulence determinants in NP and provided novel targets for the development of live attenuated vaccines and antiviral drugs against influenza viruses.IMPORTANCE The H7N9 influenza viruses that emerged in China in 2013 have caused over 1,500 human infections, with a mortality rate of nearly 40%. The viruses were initially low pathogenic but became highly pathogenic in chickens at the beginning of 2017 and caused severe disease outbreaks in poultry. Several studies suggested that the highly pathogenic H7N9 viruses have increased virulence in mammals; however, the genetic basis of the virulence of H7N9 viruses in mammals is not fully understood. Here, we found that two amino acids, 286A and 437T, in NP are prerequisites for the virulence of H7N9 viruses in mice and the mutations A286V and T437M collectively eliminate the virulence of H7N9 viruses in mice. Our study further demonstrated that the virulence of influenza viruses is a polygenic trait, and the newly identified virulence-related residues in NP may provide new targets for attenuated influenza vaccine and antiviral drug development.

SUBMITTER: Ma S

PROVIDER: S-EPMC6955278 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Amino Acid Mutations A286V and T437M in the Nucleoprotein Attenuate H7N9 Viruses in Mice.

Ma Shujie S Zhang Bo B Shi Jianzhong J Yin Xin X Wang Guangwen G Cui Pengfei P Liu Liling L Deng Guohua G Jiang Yongping Y Li Chengjun C Chen Hualan H

Journal of virology 20200106 2

The low-pathogenic H7N9 influenza viruses that emerged in 2013 acquired an insertion of four amino acids in their hemagglutinin cleavage site and thereby became highly pathogenic to chickens in 2017. Previous studies indicated that these highly pathogenic H7N9 viruses are virulent in chickens but have distinct pathotypes in mice. A/chicken/Guangdong/SD098/2017 (CK/SD098) is avirulent, with a 50% mouse lethal dose (MLD50) of >7.5 log10 50% egg infectious dose (EID50</su ...[more]

PMID: 31666373

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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.

Project description:To cross the human species barrier, influenza A viruses (IAV) of avian origin have to overcome the interferon-induced host restriction factor MxA by acquiring distinct mutations in their nucleoprotein (NP). We recently demonstrated that North American classical swine IAV are able to partially escape MxA restriction. Here we investigated whether the Eurasian avian-like swine IAV lineage currently circulating in European swine would likewise evade restriction by human MxA. We found that the NP of the influenza virus isolate A/Swine/Belzig/2/2001 (Belzig-NP) exhibits increased MxA escape, similar in extent to that with human IAV NPs. Mutational analysis revealed that the MxA escape mutations in Belzig-NP differ from the known MxA resistance cluster of the North American classical swine lineage and human-derived IAV NPs. A mouse-adapted avian IAV of the H7N7 subtype encoding Belzig-NP showed significantly greater viral growth in both MxA-expressing cells and MxA-transgenic mice than control viruses lacking the MxA escape mutations. Similarly, the growth of the recombinant Belzig virus was only marginally affected in MxA-expressing cells and MxA-transgenic mice, in contrast to that of Belzig mutant viruses lacking MxA escape mutations in the NP. Phylogenetic analysis of the Eurasian avian-like swine IAV revealed that the NP amino acids required for MxA escape were acquired successively and were maintained after their introduction. Our results suggest that the circulation of IAV in the swine population can result in the selection of NP variants with a high degree of MxA resistance, thereby increasing the zoonotic potential of these viruses.IMPORTANCE The human MxA protein efficiently blocks the replication of IAV from nonhuman species. In rare cases, however, these IAV overcome the species barrier and become pandemic. All known pandemic viruses have acquired and maintained MxA escape mutations in the viral NP and thus are not efficiently controlled by MxA. Intriguingly, partial MxA resistance can also be acquired in other hosts that express antivirally active Mx proteins, such as swine. To perform a risk assessment of IAV circulating in the European swine population, we analyzed the degree of MxA resistance of Eurasian avian-like swine IAV. Our data demonstrate that these viruses carry formerly undescribed Mx resistance mutations in the NP that mediate efficient escape from human MxA. We conclude that Eurasian avian-like swine IAV possess substantial zoonotic potential.

Dataset Information

Amino Acid Mutations A286V and T437M in the Nucleoprotein Attenuate H7N9 Viruses in Mice.

Publications

Amino Acid Mutations A286V and T437M in the Nucleoprotein Attenuate H7N9 Viruses in Mice.

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