Diversity of the H9N2 Avian Influenza Virus in Shandong Province, China

H9N2 avian influenza virus (AIV) is one of the main pathogens causing respiratory disease in chicken; however, differentiating this virus from infectious bronchitis virus (IBV) and newcastle disease virus (NDV) only using clinical signs is difficult. In this study, 492 tracheal and lung tissue samples were collected from chicken farms in Shandong reporting respiratory symptoms and tested using Reverse Transcription-Polymerase Chain Reaction (RT-PCR) for the presence of H9N2 AIVs, IBVs, and NDVs. The H9N2 AIVs positive samples were inoculated with chicken embryos. Whole-genome sequences of the positive strains were obtained using Illumina MiSeq and analyzed for genetic evolution and key amino acid sites mutation. Seventy-two samples were positive for H9N2 subtype AIV, with a positive rate of 14.63%, while the positive rates of IBV and NDV were 6.10% and 0.41%, respectively. Thirty-four strains of H9N2 AIVs were obtained from positive samples. Phylogenetic tree analysis of HA and NA genes revealed that the 34 H9N2 AIV strains belonged to Y280-like and F/98-like branches, respectively. Clear temporalphylogenetic branching was observed, with some strains found in the "pre-2013 isolates" clade and others in the "post-2013 isolates" clade, which raised the possibility that strains in the former clade may have undergone recombination with viral strains from 10 years ago. Among the internal amino acid sites that are key to mammalian adaptation, all strains had an I368V mutation in the PB1 gene that enhanced viral transmissibility in mammals, and the PB2 genes of some strains were mutated to enhance the mammalian adaptation of I292V and A588V. Thus, the H9N2 AIV gene segments in Shandong have different degrees of recombination and gene variation, necessitating vigilant monitoring of virus variation.