Isolation,Identification And Genetic Evolution Analysis Of H9N2 Avian Influenza In Part Of Hubei Province

Avian Influenza Virus(AIV)has emerged as a significant threat in both wild birds and poultry since its emergence.Since 2016,the isolation rate of H9N2 subtype AIV has gradually increased and has become the most prevalent AIV subtype in China,with the Y280 lineage being the dominant one.Some strains of the H9N2 subtype AIV have gradually acquired the ability to bind to α-2,6 sialic acid receptors,which poses a risk of infection to humans and is subject to widespread variation and recombination,which can provide internal genes for other subtypes of AIV,posing a great threat to the poultry industry and public health safety.Currently used inactivated vaccines for the H9N2 subtype AIV do not effectively control the spread and variation of H9N2 subtype AIV in chicken flocks.Monitoring the prevalence and genetic variation of the H9N2 subtype AIV is of great significance for the prevention and control of AIV.This study isolated and identified the H9N2 subtype AIV in poultry in Hubei Province and conducted a whole-genome genetic evolution analysis,providing a theoretical basis for the genetic variation patterns of AIV in Hubei Province.The main research results are as follows:1.Isolation,identification and purification of H9N2 subtype AIVTo isolate and identify H9N2 subtype AIV,a total of 341 throat swabs and cloacal swabs have been collected from poultry at various locations within Hubei Province in 2022.These locations included farmers’ markets,live poultry trading markets,slaughterhouses,and farms.The isolation rate for the virus is 5.3%.Eighteen strains of H9N2 subtype AIV have been successfully isolated and identified,with seventeen strains originating from chickens and one from ducks.The virus purification is completed by the chicken embryo limited dilution method.2.Whole genome sequencing and genetic evolution analysis of isolatesBy using the RT-PCR method,the isolated strains’ whole genomes were amplified and sequenced for analysis.Genetic evolution analysis was carried out on the whole genome,exploring the prevalence and variation of H9N2 subtype AIV in Hubei Province at the molecular level.The maximum likelihood method was used to construct the genetic evolution tree of eight gene segments(HA,NA,PB2,PB1,PA,NP,M,and NS)of the H9N2 subtype avian influenza isolates.The results showed that the isolates were triple reassortant H9N2 subtype AIV,with the HA and NA segments belonging to the Y280 lineage,the PB2 and M segments belonging to the G1 lineage,and the PA,PB1,NP,and NS segments belonging to the F98 lineage.Genetic evolution and homology analysis showed that the isolates had high homology with human-origin AIVs,with the HA and NA segments having high homology with the A/Fujiansiming/19/2021(H9N2)strain,and the six internal gene segments having high homology with the A/Beijing/1/2017(H9N2),A/China/CSKFQ-22-5/2022(H3N8),A/China/ZMD-22-2/2022(H3N8)strains.The M gene of isolate A/duck/Hubei/195/2022(H9N2)had a distant genetic distance from the other seventeen isolates.3.Key sites and biological characteristics analysis of isolatesAll 18 H9N2 subtype avian influenza isolates were low pathogenicity avian influenza strains,and the HA protein cleavage site was PSRSSR↓GLF for all isolates.The glycosylation site of the HA protein did not change,and the NA segment of the isolates had novel variations in the red blood cell binding site and antigenic determinant cluster site.Of the eighteen isolates,six retained the 627 E site on the PB2 protein,while twelve had a mutation at the 627 V site.Analysis of the adamantane-resistant sites related to the M2 protein showed that the isolates had adamantane resistance at the molecular level.Biological characteristic tests showed that only one H9N2 subtype isolate,A/duck/Hubei/195/2022(H9N2),could proliferate on A549 cells.In summary,this study isolated and identified 18 strains of H9N2 subtype avian influenza viruses(AIVs),completed the whole-genome sequencing,genetic evolution,and variation analysis of the isolated strains.The study revealed the epidemic pattern of H9N2 subtype AIV in poultry in Hubei Province,providing support for the prevention and control of avian influenza.