Genetic evolution and substitution frequency of avian influenza virus HA gene in chicken H9N2 subtype in China in the last 20 years

Low pathogenic avian influenza (LPAI) H9N2 subtype virus has been prevalent in domestic poultry in China over two decades. This study was to determine the genetic evolution trend of H9N2 avian influenza virus (AIV) under immune pressure of vaccine.H9 HA sequences of 40 isolates from the present study and 136 pandemic strains and 7 classical strains from China downloaded from GenBank, were genetically analyzed to determine evolution, molecular characteristic, and mutation frequency.Phylogenetic trees analysis suggested that H9N2 subtypes AIV could be clustered into 5 distinct lineages: G1-like, BJ94-like, Y280-like, S2-like and Americans lineages. Most H9N2 isolates in 2005-2014 belonged to S2-like sub-genotype, suggesting that this genotype was the dominate isolates in China. Further more, comparison based on the amino acid sequence showed that different lineages have their distinct characteristics, and significant accumulations of amino acid variation were also found. In addition, in comparison with reference Ck/BJ/1/1994 HA gene, average annual substitution rates of H9N2 pandemic strain nucleotide and amino acid were 5.73 x 10⁻³ and 4.25 x 10⁻³ from 1994 to 2014, respectively. Substitution rate during 2011-2014 were 6.35 x 10⁻³ and 5.32 x 10⁻³, higher than that during the period of 2006-2010 (5.22 x 10⁻³ and 3.70 x 10⁻³) and even much higher than that during the 1999-2005 (0.74 x 10⁻³ and 0.50 x 10⁻³), when the vaccines were initially applied in the field.Overall, these data indicate that the mismatch between H9N2 vaccine strains and pandemic strains drives the virus to quickly mutate.