Forty-two H9N2 subtype AIV strains were isolated from vaccinated commercial chickens in China from 2012 to 2015. Their HA genes had nucleotide sequence homology from 86.7% to 99.7%, and similarity to the classic vaccine strain was 88.6%–92.6%. A comparison was carried out with published HA genes (410 H9 strains) and whole genomes (306 strains) isolated in China during 2012–2015. Interestingly, 99.1% (448/452) of Chinese H9N2 AIV belonged to lineage h9.4.2, and 98.5% (445/452) of the viruses belonged to h126.96.36.199. Meanwhile, 99.6% (443/445) of lineage 188.8.131.52 viruses had PSRSSR↓GLF instead of PARSSR↓GLF motifs in the HA cleavage sites; 98.2% (444/452) of HA genes showed human receptor binding associated mutation Q226L. A total of 96.8% (337/348) of the viruses had three amino-acid deletions at 63–65 in the NA stalk, associated with enhanced virulence in chickens and mice; 97.1% (338/348) of M2 proteins had the S31N mutation associated with adamantane resistance in humans. Two H9 viruses isolated in this study were highly homologous to the human-origin H9N2 virus reported in 2013. The isolates were divided into four different genotypes (U, S, V, and W). Genotype S was the major one, accounting for 94.8% (330/348). Genotypes V and W were new reassortment genotypes, with genotype W recombined with the PB2 gene originating from the new wild waterfowl-like lineage. According to the cross-HI antibody titer data, HA gene evolution, and isolation history, the isolates were divided into A, B, and C antigenic groups successively. All the antigenic group viruses were found to circulate throughout China. This study emphasizes the importance of updated vaccine and strengthened veterinary biosecurity on poultry farms and trade markets.
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