Abstract
Novel avian H7N9 virus emerged in China in 2013 resulting in a case fatality rate of around 39% and continues to pose zoonotic and pandemic risk. Amino acid substitutions in PB2 protein were shown to influence the pathogenicity and transmissibility of H7N9 following experimental infection of ferrets and mice. In this study, we evaluated the role of amino acid substitution PB2-627K or compensatory changes at PB2-591K and PB2-701N, on the tropism and replication competence of H7N9 viruses for human and swine respiratory tracts using ex vivo organ explant cultures. Recombinant viruses of A/Shanghai/2/2013 (rgH7N9) and its mutants with PB2-K627E, PB2-K627E + Q591K and PB2-K627E + D701N were generated by plasmid-based reverse genetics. PB2-E627K was essential for efficient replication of rgH7N9 in ex vivo cultures of human and swine respiratory tracts. Mutant rgPB2-K627E + D701N replicated better than rgPB2-K627E in human lung but not as well as rgH7N9 virus. The rgPB2-K627E mutant failed to replicate in human type I-like pneumocytes (ATI) and peripheral blood monocyte-derived macrophages (PMϕ) at 37 °C while the compensatory mutant rgPB2-K627E + Q591K and rgPB2-K627E + D701N had partly restored replication competence in PMϕ. Our results demonstrate that PB2-E627K was important for efficient replication of influenza H7N9 in both human and swine respiratory tracts.
Highlights
Novel avian H7N9 virus emerged in China in 2013 resulting in a case fatality rate of around 39% and continues to pose zoonotic and pandemic risk
In the avian DF-1 cells, rgPB2-K627E demonstrated its temperature sensitive phenotype and replicated poorly at 33 °C (Fig. 1A) while the mutant viruses with the human compensatory markers, rgPB2-K627E +Q591K and rgPB2K627E +D701N were able to replicate to similar titers as rgH7N9 which has PB2-627K (Fig. 1A)
We evaluated the effects of PB2-E627K mutation as well as two mammalian adaptation markers, PB2-Q591K and D701N on H7N9 virus replication competence in ex vivo cultures of human and swine respiratory tracts and in vitro cultures of human primary ATI and PMφ.While avian H7N9 viruses invariably have PB2627E, A/Shanghai/2/2013 (Sh2) (H7N9) virus which was isolated from a patient with severe disease had acquired the mammalian adaptation mutation PB2-E627K, and this is reflected in recombinant rgH7N9 virus used in this study
Summary
Novel avian H7N9 virus emerged in China in 2013 resulting in a case fatality rate of around 39% and continues to pose zoonotic and pandemic risk. We evaluated the role of amino acid substitution PB2-627K or compensatory changes at PB2-591K and PB2-701N, on the tropism and replication competence of H7N9 viruses for human and swine respiratory tracts using ex vivo organ explant cultures. Deletion of amino acids in the stalk region of the NA protein (at position 69 to 73) was previously found in HPAI H5N1 virus and is an adaptation of influenza viruses to replication in terrestrial poultry such as chicken and may affect viral replication efficiency and tissue tropism in the respiratory tract[4]. Similar studies have been conducted using swine respiratory tissue explant cultures to address the susceptibility of the swine respiratory tract to H7N9 viruses[20] This is relevant since swine have long been recognized as a potential “mixing vessel” of human and avian influenza viruses[21,22], together with its critical role in the emergence of pandemic H1N1 in 200923. Given the close contact between pigs, poultry and humans in agricultural settings in China, it is important to investigate the impact of genetic adaptations of H7N9 viruses in swine
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