Genetic Evolution And Biological Characteristics Of 12avian Influenza Virus Strains H9N2 Subtype In Poultry

H9N2 subtype avian influenza virus(AIV)is widely prevalent in China.In recent years,H9N2 AIV has not only caused serious impact on poultry breeding industry in China,but also can cross the host species barrier via gene recombination to cause infection of mammals and humans,thus posing a certain threat to public health.In this study,12 H9N2 subtype AIV strains isolated and identified in Shandong and Henan province from 2020 to 2021 were selected to study their genetic evolution,antigenic variation and pathogenicity to mice.First,RT-PCR amplification and whole genome sequencing were carried out for 12 purified H9N2 AIV strains,and the genetic evolution of each gene fragment and variations of important amino acid residues were analyzed based on the viral genome.The results showed that all 8 gene fragments of 12 H9N2 AIV strains belonged to Eurasian lineage,but the molecular evolutionary relationships of 8 gene fragments were different.Among them,HA gene and NA genes were from Y280-like.M gene and NP gene of 10 isolates were from G1-like and F98-like respectively,while the remian 2isolates were from Y439-like.In terms of PA and NS genes,there were 9 isolates were from F98-like and 3 isolates from Y439-Like,respectively.PB1 genes were all from F98-like and PB2 genes were all from G9-like.These results indicated that 12 H9N2 AIV strains came from different evolutionary routes.Analysis of the key amino acid sites of 12 H9N2 AIV strains showed that the receptor binding sites 149,198,234 and 235 of HA protein had the most variations.Among them,the amino acid at position 234(amino acid 226 of H3 numbering)was switched to L in 9 isolates,S in 2 isolates and V in 1 isolates.The amino acid at position 235 was switched to M in all isolates.Potential glycosylation site at residue 218?220 was found in HA protein in 3isolates,and loss of a potential glycosylation site at residue 492?494 was found in all of the isolates.The cleavage sites of HA gene were all PSRSSR?GLF,which accorded with the characteristics of low pathogenic AIV.Among the 12 H9N2 AIV strains,there were 6 strains possessed or lost the glycosylation site at residue 261?263 of NA protein respectively.The potential glycosylation site at residue 399?401 of NA protein was lost in all isolates.Amino acids in the neck of NA protein of all strains were absent,which was consistent with the variation characteristics of H9N2 AIV epidemic strains in China in recent years.The amino acid at sites 224,356,615 of PA protein were mutate in 3 isolates,and the amino acid at site237 was mutate in 1 isolate.The amino acid at position 672 was switched to L in all isolates.The mutation of M2 protein in 12 H9N2 AIV strains was found in 1 isolate at sites 27 and A30T,respectively,The amino acid at site 28 of M2 protein was mutate in 2 isolates.The amino acid at site 31 of M2 protein,only 3 isolates were S and the remian 9 isolates all switched to N.The amino acid at sites 103,127,205 of NS1 protein showed mutation in 3isolates,and the 106 residue site of NS1 protein was found mutant in 4 isolates.The amino acid at site 34 of NP protein was found mutant in 2 isolates.I292V mutant of PB2 protein was found in all 12 strains while the amino acid at site 627 was E and E627K was not found in all strains.The above results indicated that some key amino acid sites of 12 H9N2 AIV strains were varied to different degrees.Cross hemagglutination inhibition test was performed to analyze the antigenic variation of the 12 H9N2 AIV strains.The results showed that there were great differences in the titer of cross HI and R value among the 12 H9N2 AIV strains,and the cross HI and R value between 3 isolates and the other 9 isolates were all 0,indicating that the antigenic difference of H9N2 AIV in Shandong and its surrounding areas became more and more significant during the evolution of H9N2 AIV in recent two years.The biological activity of 12 strains of H9N2 AIV was determined,and the results showed that 12 strains of H9N2 AIV had good adaptability to SPF chicken embryos,and the EID₅₀value ranged from 10^7.17to 10^8.63.However,the values of TCID₅₀varied from 10^0.5to10^7.5TCID₅₀/0.2ml in MDCK and from 10^0.5to 10^7.5TCID₅₀/0.2ml in A549 cells respectively.BALB/c mice were infected intranasally with 10~6EID₅₀of H9N2 AIV in a 50-u L volume,and it was found that 6 of the 12 H9N2 AIV strains could replicate well in the lungs of mice without adaptation.The infection of some strains affected the normal growth,weight loss and even caused death of mice.These results indicate that there are differences in the replication ability of H9N2 AIV strains in mammalian cells and in vivo in recent two years.Therefore,attention should be paid to the changes of pathogenicity of H9N2 AIV in poultry to mammals,and provide early warning for cross-species infection of low pathogenicity AIV in mammals.