Analysis of the phylogeny of Chinese H9N2 avian influenza viruses and their pathogenicity in mice

We isolated nineteen strains of H9N2 influenza virus from farms across five northern Chinese provinces between 2001 and 2012. Sequence analysis of the genes for the two surface glycoproteins revealed that residue 226 of the hemagglutinin (HA) of eight isolates was a leucine. A T300I mutation in three strains resulted in the loss of a potential glycosylation site. The P315S mutation in seven strains added a potential glycosylation site in HA. The isolates CK/HN/323/08 and CK/HN/321/08 had a full-length neuraminidase (NA) that differed from those seen in other isolates. Phylogenetic and molecular analysis revealed that the nineteen strains shared common ancestry with strains BJ/94 and G1. We examined eight gene sequences in the present study and concluded that the HA and NS genes appeared to be derived directly from BJ/94. The remaining six genes evolved from different reference strains. Specifically, the NA and PA genes of CK/HN/321/08 and CK/HN/323/08 clustered with the G9 and Y439 branch, respectively, and the PB2 genes of CK/SD/513/11 and CK/GS/419/12 were in an unknown lineage. We found evidence that seven new genotypes had undergone intra-subtype reassortment. A mouse infection experiment with six selected isolates showed that five of these isolates were able to replicate in mouse lungs without adaptation. Viral replication in infected mice resulted in minimal weight loss, suggesting that these H9N2 avian influenza viruses had low virulence in mammals. Our findings highlight the genetic and biological diversity of H9N2 avian influenza viruses circulating in China and emphasize the importance in continuing surveillance of these viruses so as to better understand the potential risks they pose to humans.