Analyses On Evolution Of H9N2Avian Influenza Viruses From Five Provinces In Northern China During2001-2012and Their Pathogenicity In Mice

H9N2influenza viruses were firstly isolated from chickens and pigs from Guangdong and Hong Kong in1994and1998respectively. In1999, H9N2avian influenza viruses infected2humans in Hong Kong suggesting H9N2viruses not only could cause a huge loss for poultry farm but also could pose severe threat to public health. However, the mechanisms for H9N2influenza virus to transmit in different species are still unknown. In this study,19strains of H9N2avian influenza viruses isolated from chickens from five provinces in Northern China from2001to2012were intensively analyzed. At the same time,6strains viruses were infected mice and evaluated pathogenicity of mammals.We isolated nineteen strains of H9N2influenza virus from farms across five Northern Chinese provinces between2001and2012. Sequences analysis of two surface glycoproteins revealed that residue226in eight HA was a leucine, which prefers binding the human a-2,6sialic acid receptor and poses a threat to human. A T300I mutation in three strains resulted in the loss of a potential glycoprotein site. The P315S mutation in seven strains added a potential glycoprotein site in HA, potential glycoprotein have been thought about the virus host range and virulence. CK/HN/321/08and CK/HN/323/08strains had a full-length NA that differed from those seen in other isolates. The previous research found this three-amino-acid absence help the virus release from infected cells. Phylogenetic and molecular analyses revealed that nineteen strains shared common ancestry with strains BJ/94and G1. We examined eight gene sequences in the present study and concluded that HA and NS genes appeared to be directly derived from BJ/94. The remaining five genes evolved from different reference strains. Particularly, NA and PA genes of CK/HN/321/08and CK/HN/323/08clustered with the G9and Y439branch, respectively, and PB2genes of CK/SD/513/11and CK/GS/419/12were in an unknown lineage. We identified that seven new genotypes underwent intra-subtype reassortment compared with the previous studies.According to the data of molecular evolution,6strains viruses that had different antigenic sites in HA were selected for preparing their special antisera. Cross HI results indicated that the antigenicity had the difference in six H9N2strains.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 the virulence of H9N2avian influenza viruses had a slight effect on mammal.In summary,19H9N2viruses isolated from five provinces in northern China from2001to2012were studied, and results showed that all of viruses were derived from H9N2avian influenza viruses isolated in the early period in China, which have multiple genotypes and obvious diversity of biological properties. Some H9N2viruses can replicate in mouse lung and pose a threat tomammals.