title: Assessment of the internal genes of influenza A (H7N9) virus contributing to high pathogenicity in mice.
abstract: The recently identified H7N9 influenza A virus has caused severe economic losses and worldwide public concern. Genetic analysis indicates that its six internal genes all originated from H9N2 viruses. However, the H7N9 virus is more highly pathogenic in humans than H9N2, which suggests that the internal genes of H7N9 have mutated. To analyze which H7N9 virus internal genes contribute to its high pathogenicity, a series of reassortants was generated by reverse genetics, with each virus containing a single internal gene of the typical A/Anhui/1/2013 (H7N9) (AH-H7N9) virus in the genetic background of the A/chicken/Shandong/lx1023/2007 (H9N2) virus. The replication ability, polymerase activity, and pathogenicity of these viruses were then evaluated in vitro and in vivo. These recombinants displayed high genetic compatibility, and the H7N9-derived PB2, M, and NP genes were identified as the virulence genes for the reassortants in mice. Further investigation confirmed that the PB2 K627 residue is critical for the high pathogenicity of the H7N9 virus and the reassortant containing the H7N9-derived PB2 segment (H9N2-AH/PB2). Notably, the H7N9-derived PB2 gene displayed greater compatibility with the H9N2 genome than that of H7N9, endowing the H9N2-AH/PB2 reassortant with greater viability and virulence than the parental H7N9 virus. In addition, the H7N9 virus, with the exception of the H9N2 reassortants, could effectively replicate in human A549 cells. Our results indicate that PB2, M, and NP are the key virulence genes, together with the surface hemagglutinin (HA) and neuraminidase (NA) proteins, contributing to the high infectivity of the H7N9 virus in humans.
  
  
  OBJECTIVE
  To date, the novel H7N9 influenza A virus has caused 437 human infections, with approximately 30% mortality. Previous work has primarily focused on the two viral surface proteins, HA and NA, but the contribution of the six internal genes to the high pathogenicity of H7N9 has not been systematically studied. Here, the H9N2 virus was used as a genetic backbone to evaluate the virulence genes of H7N9 virus in vitro and in vivo. Our data indicate that the PB2, M, and NP genes play important roles in viral infection in mice and, together with HA and NA, contribute to the high infectivity of the H7N9 virus in humans.
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Yuhai
authors.initials: Y
authors.lastname: Bi
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Department of Clinical Laboratory, Beijing Shijitan Hospital, Capital Medical University, Beijing, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Xie
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Shuang
authors.initials: S
authors.lastname: Zhang
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Li
authors.affiliation: Laboratory of Protein Engineering and Vaccines, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
authors.forename: Haixia
authors.initials: H
authors.lastname: Xiao
authors.affiliation: BGI-Shenzhen, Shenzhen, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Jin
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Weinan
authors.initials: W
authors.lastname: Zheng
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: Jia
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Sun
authors.affiliation: Key Laboratory of Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: Jinhua
authors.initials: J
authors.lastname: Liu
authors.affiliation: Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences and Comparative Medicine Center, Peking Union Medical College, Key Laboratory of Human Disease Comparative Medicine, Ministry of Health, Beijing, China.
authors.forename: Chuan
authors.initials: C
authors.lastname: Qin
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Laboratory of Protein Engineering and Vaccines, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China Office of Director General, Chinese Center for Disease Control and Prevention, Beijing, China Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, Hanzhou, China Gaof@im.ac.cn liuwj@im.ac.cn.
authors.forename: George F
authors.initials: GF
authors.lastname: Gao
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Gaof@im.ac.cn liuwj@im.ac.cn.
authors.forename: Wenjun
authors.initials: W
authors.lastname: Liu
mesh_terms: D000818:Animals; D002460:Cell Line; D002642:Chick Embryo; D004195:Disease Models, Animal; D004847:Epithelial Cells / Q000821:virology; D005260:Female; D006801:Humans; D064766:Influenza A Virus, H7N9 Subtype / Q000235:genetics* / Q000472:pathogenicity* / Q000502:physiology; D053127:Influenza A Virus, H9N2 Subtype / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology; D008807:Mice, Inbred BALB C; D009976:Orthomyxoviridae Infections / Q000473:pathology* / Q000821:virology*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism; D016865:Reassortant Viruses / Q000235:genetics / Q000472:pathogenicity; D059386:Reverse Genetics; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics* / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D037521:Virulence Factors; D012324:RNA-Dependent RNA Polymerase