Influenza A/H7N9 virus infection of human and swine respiratory epithelium : tissue tropism and pathogenesis

Abstract

Since February 2013, a novel subtype of avian-origin influenza H7N9 virus has emerged in Eastern China and has now become enzootic in Mainland China. Majority of the infected patients were suffered from acute respiratory distress syndrome (ARDS) with a case fatality rate of approximately 39%. Genetic analysis revealed that human H7N9 isolates possessed several key mutations, including substitutions in haemagglutinin (HA) and polymerase basic protein 2 (PB2) genes. The HA protein of H7N9 isolates possesses T160A and Q226L (H3 numbering) which enhances the receptor binding specificity towards α2,6 sialic acid receptors and the presence of PB2-E627K is associated with enhanced virulence in mice. My project aims to study the pathogenicity of the novel influenza A/H7N9 virus and investigate the important viral determinants contributing to the pathogenesis of H7N9 virus infection. Several risk assessment models including ex vivo cultures of human and swine respiratory tracts, in vitro cultures of human type I-like pneumocytes (ATI) and peripheral blood monocyte-derived macrophages (PMϕ) and plasmid-based reverse genetics were employed. The pathogenesis of H7N9 viruses was compared with the highly pathogenic avian influenza (HPAI) H5N1 virus and the 2009 pandemic H1N1 (H1N1pdm) virus. Influenza H7N9 viruses extensively infected the ex vivo cultures of human bronchus and lung. The extent of infection was greater than the HPAI H5N1 ex vivo. H7N9 viruses were found to be a moderate inducer of pro-inflammatory cytokines and chemokines in primary in vitro cell cultures, whereas HPAI H5N1 was a potent inducer and H1N1pdm was a comparatively poorer inducer. Recombinant viruses with loss-of-function mutations in HA and PB2 genes were constructed. HA-Q226L was crucial for the tissue tropism of H7N9 viruses in the human and swine upper respiratory tract. PB2-K627E mutation abolished the infection of H7N9 viruses in human and swine respiratory tracts. The introduction of compensatory mammalian markers, PB2-Q591K or D701N only partially rescued the replication efficiency. Besides, human isolates from the first and the second wave of H7N9 outbreak were compared. Six Hong Kong H7N9 human isolates from the second wave of H7N9 outbreak including two non-fatal infections and four fatal infections were compared with an isolate from a fatal case of the first wave of H7N9 outbreak, A/Shanghai/2/2013. Several adaptive mutations were identified in the second wave H7N9 viruses, such as HA-A135T and Polymerase acidic protein (PA)-L336M, which were known to enhance HA stability and increase polymerase activity and pathogenicity in mice, respectively. Efficient viral replication was found in human respiratory ex vivo culture explants infected with H7N9 viruses. H7N9 virus is a moderate pro-inflammatory cytokine and chemokine inducer when compared with H5N1 and H1N1pdm. HA-Q226L and PB2-E627K were the key molecular determinants for infection in human and swine respiratory tract ex vivo cultures. Although no further key mammalian adaptation markers were observed in the second wave human H7N9 isolates, the pandemic risk of influenza H7N9 virus can never be underestimated since it can gain new adaptive features through evolution. This study elucidated the molecular determinants associated with acquiring human-to-human transmission potential that is crucial for pandemic preparedness.