H9N2 subtype avian influenza virus (AIV) is widely prevalent in poultry, and the virus is becoming adaptive to mammals, which poses pandemic importance. Here, BALB/c mice were employed as a model to evaluate the adaption in mammals of 21 field H9N2 viruses isolated from avian species between 2016 to 2019 in China. The replication capacity of the viruses was evaluated in the lungs of mice. The pathogenicity of the viruses were compared by weight loss and lung lesions from infected mice. The whole genomic sequences of the viruses were further characterized to define the associated phenotypes of the H9N2 viruses in vitro and in vivo. The results showed that most viruses could replicate well and cause lesions in the mouse lungs. The propagation capacity in MDCK cells and damage to respiratory tissues of the infected mice corresponded to relative viral titers in the mouse lungs. Further genome analysis showed that all of the H9N2 viruses belonged to the same genotype, G57, and contained a couple of amino acid substitutions or deletions that have been demonstrated as avian-human markers. Additionally, nine amino acids residues in seven viral proteins were found to be correlated with the replication phenotypes of the H9N2 viruses in mammals. The study demonstrated that a well-defined H9N2 AIV genotype with high adaption in mammals was prevalent in China in recent years. Further investigations on the role of the identified residues and continuous surveillance of newly identified mutations associated with host adaption should be strengthened to prevent any devastating human influenza pandemics.
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