Sequence Analysis And Pathogenicity Of H9N2 Subtype Avian Influenza Virus Isolates

H9N2 subtype Avian influenza virus(AIV)seriously affects the health development of the poultry industry in China,and also poses a threat to human public health safety.It is of great significance to strengthen the epidemiological investigation,virus isolation,identification and genetic evolution analysis of H9N2 subtype AIV.In this study,an epidemiological survey of H9 subtype AIV was conducted in the vaccinated farms in seven different provinces of China from 2021 to 2022.Six samples and two samples kept in the laboratory were collected for virus isolation,identification,whole genome sequencing and genetic evolution analysis.The antigenic differences were analyzed by preparation of specific immune serum and cross-hemagglutination inhibition test.Two isolates from different small branches and different avian sources were selected for pathogenicity test on SPF chickens.1.A total of 433 samples were collected,and the total positive rate of H9 was6.70%.The positive rate of H9 in chickens was higher than that in waterfowl,and the detection rate in winter was higher than that in summer and autumn.2.Eight H9N2 strains were isolated and identified,including 2 waterfowl strains and 6 chicken strains.All the eight AIV strains belonged to H9.4.2.5 sub-branch of Y280 subgroup of Eurasian branch.The 234 L mutation in the HA gene of the eight isolates suggested that the isolates had the characteristics of binding to mammalian sialic acid receptors.There were 7-10 potential N-linked glycosylation sites(PNLGSs)in the HA gene,and two of them had new PNLGSs.The shortened neck of NA in all 8isolates resulted in increased pathogenicity.Some isolates increased PNLGSs at 368,which may have an important effect on the inhibition of NA by serum antibodies.Phylogenetic analysis showed that all the 8 strains were recombinant viruses,and even recombined with different subtypes of AIV.The analysis of important amino acid sites showed that all the isolates had mutations that enhanced replication and pathogenicity in mammals.3.The H9N2 subtype AIV strains isolated from different branches showed antigenic differences.Two of the isolates were pathogenic to SPF chicken embryos and SPF chickens,and both of them could cause infection of healthy chickens in the same group.The infected SPF chickens could shed virus in the respiratory and digestive tracts,and the challenged SPF chickens could produce good humoral immunity.Therefore,the monitoring of H9N2 subtype AIV in poultry farms should be strengthened,and the genetic evolution analysis of the constantly emerging new strains should be carried out to provide molecular biological basis for the virulence enhancement,cross-species transmission,molecular evolution and antigenic variation of AIV in the influenza virus ecosystem,and also provide basis for the renewal of the seed virus pool of vaccine manufacturers.To put forward epidemic prevention ideas for the prevention and control of H9N2 infection in livestock farms.