M Gene Reassortment in H9N2 Influenza Virus Promotes Early Infection and Replication: Contribution to Rising Virus Prevalence in Chickens in China.
Segment reassortment and base mutagenesis of influenza A viruses are the primary routes to the rapid evolution of high-fitness virus genotypes. We recently described a predominant G57 genotype of avian H9N2 viruses that caused countrywide outbreaks in chickens in China during 2010 to 2013, which led to the zoonotic emergence of H7N9 viruses. One of the key features of the G57 genotype is the replacement of the earlier A/chicken/Beijing/1/1994 (BJ/94)-like M gene with the A/quail/Hong Kong/G1/1997 (G1)-like M gene of quail origin. We report here the functional significance of the G1-like M gene in H9N2 viruses in conferring increased infection severity and infectivity in primary chicken embryonic fibroblasts and chickens. H9N2 virus housing the G1-like M gene, in place of the BJ/94-like M gene, showed an early surge in viral mRNA and viral RNA (vRNA) transcription that was associated with enhanced viral protein production and with an early elevated release of progeny virus comprising largely spherical rather than filamentous virions. Importantly, H9N2 virus with the G1-like M gene conferred extrapulmonary virus spread in chickens. Five highly represented signature amino acid residues (37A, 95K, 224N, and 242N in the M1 protein and 21G in the M2 protein) encoded by the prevalent G1-like M gene were demonstrated to be prime contributors to enhanced infectivity. Therefore, the genetic evolution of the M gene in H9N2 virus increases reproductive virus fitness, indicating its contribution to the rising virus prevalence in chickens in China.IMPORTANCEWe recently described the circulation of a dominant genotype (genotype G57) of H9N2 viruses in countrywide outbreaks in chickens in China, which was responsible, through reassortment, for the emergence of H7N9 viruses that cause severe human infections. A key feature of the genotype G57 H9N2 virus is the presence of the quail-origin G1-like M gene, which had replaced the earlier BJ/94-like M gene. We found that H9N2 virus with the G1-like M gene, but not the BJ/94-like M gene, showed an early surge in progeny virus production and more severe pathology and extrapulmonary virus spread in chickens. Five highly represented amino acid residues in the M1 and M2 proteins derived from the G1-like M gene were shown to mediate enhanced virus infectivity. These observations enhance what we currently know about the roles of reassortment and mutations in virus fitness and have implications for assessing the potential of variant influenza viruses that can cause a rising prevalence in chickens.
Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, and State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, China.
Juan
J
Pu
Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, and State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, China.
Honglei
H
Sun
Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, and State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, China.
Yi
Y
Qu
Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, and State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, China.
Chenxi
C
Wang
Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, and State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, China.
Weihua
W
Gao
Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, and State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, China.
Junda
J
Zhu
Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, and State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, China.
Yipeng
Y
Sun
CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Yuhai
Y
Bi
Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, and State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, China.
Yinhua
Y
Huang
School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Loughborough, United Kingdom.
Kin-Chow
KC
Chang
Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
Jie
http://orcid.org/0000-0001-8176-9951
J
Cui
Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, and State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, China ljh@cau.edu.cn.
Jinhua
J
Liu
D000818:Animals; D002645:Chickens; D004252:DNA Mutational Analysis; D005347:Fibroblasts / Q000821:virology*; D053127:Influenza A Virus, H9N2 Subtype / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology* / Q000648:ultrastructure; D005585:Influenza in Birds / Q000473:pathology* / Q000821:virology; D016865:Reassortant Viruses / Q000235:genetics / Q000502:physiology*; D014763:Viral Matrix Proteins / Q000235:genetics*; D014771:Virion / Q000648:ultrastructure; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics*; D014779:Virus Replication
C031540:M-protein, influenza virus; D014763:Viral Matrix Proteins; D037521:Virulence Factors
H9N2 virus; M gene; chicken; reassortment; replication