title: Amino acid substitutions in polymerase basic protein 2 gene contribute to the pathogenicity of the novel A/H7N9 influenza virus in mammalian hosts.
abstract: A novel avian-origin influenza A/H7N9 virus emerged in 2013 to cause more than 130 cases of zoonotic human disease, with an overall case fatality rate of around 30% in cases detected. It has been shown that an E-to-K amino acid change at residue 627 of polymerase basic protein 2 (PB2) occurred frequently in the H7N9 isolates obtained from humans but not in viruses isolated from poultry. Although this mutation has been reported to confer increased mammalian pathogenicity in other avian influenza subtypes, it has not been experimentally investigated in the H7N9 virus. In this study, we determined the contribution of PB2-E627K in H7N9 virus to its pathogenicity in mammalian hosts. In addition, the compensatory role of the PB2 mutations T271A, Q591K, and D701N in H7N9 virus was investigated. We characterized the activity of polymerase complexes with these PB2 mutations and found that they enhance the polymerase activity in human 293T cells. The rescued mutants enhanced growth in mammalian cells in vitro. Mice infected with the H7N9 mutant containing the avian signature protein PB2-627E showed a marked decrease in disease severity (weight loss) and pathology compared to mice infected with the wild-type strain (PB2-627K) or other PB2 mutants. Also, mutants with PB2-627E showed lower virus replication and proinflammatory cytokine responses in the lungs of the virus-infected mice, which may contribute to pathogenicity. Our results suggest that these amino acid substitutions contribute to mouse pathogenicity and mammalian adaptation.
  
  
  OBJECTIVE
  A novel avian H7N9 influenza A virus emerged in east China in 2013 to cause zoonotic human disease associated with significant mortality. It is important to understand the viral genetic markers of mammalian adaptation and disease severity in this H7N9 virus. Since many human (but not avian) H7N9 virus isolates have an amino acid substitution at position E627K in the polymerase basic protein 2 (PB2) gene, we investigated the role of this and other functionally related mutations for polymerase activity in vitro, virus replication competence, and pathogenicity in the mouse model. We found that E627K and functionally related mutations are associated with increased polymerase activity, increased viral replication competence, and increased disease severity in mice.
authors.affiliation: Centre of Influenza Research, School of Public Health, HKU Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong.
authors.forename: Chris Ka Pun
authors.initials: CK
authors.lastname: Mok
authors.forename: Horace Hok Yeung
authors.initials: HH
authors.lastname: Lee
authors.forename: Maxime
authors.initials: M
authors.lastname: Lestra
authors.forename: John Malcolm
authors.initials: JM
authors.lastname: Nicholls
authors.forename: Michael Chi Wai
authors.initials: MC
authors.lastname: Chan
authors.forename: Sin Fun
authors.initials: SF
authors.lastname: Sia
authors.forename: Huachen
authors.initials: H
authors.lastname: Zhu
authors.forename: Leo Lit Man
authors.initials: LL
authors.lastname: Poon
authors.forename: Yi
authors.initials: Y
authors.lastname: Guan
authors.forename: Joseph Sriyal Malik
authors.initials: JS
authors.lastname: Peiris
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002645:Chickens; D016207:Cytokines / Q000235:genetics / Q000276:immunology; D005260:Female; D006801:Humans; D064766:Influenza A Virus, H7N9 Subtype / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity*; D005585:Influenza in Birds / Q000235:genetics / Q000276:immunology / Q000821:virology*; D007251:Influenza, Human / Q000235:genetics / Q000276:immunology / Q000821:virology*; D051379:Mice; D008807:Mice, Inbred BALB C; D020125:Mutation, Missense; D011201:Poultry Diseases / Q000235:genetics / Q000276:immunology / Q000821:virology*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014774:Virulence
chemical_list: D016207:Cytokines; C057027:PB2 protein, Influenzavirus A; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase