Epidemiological Investigation And Pathogenicity Of H9N2 Avian Influenza In Chickens In Shandong Province

Avian Influenza（AI）is a highly contact respiratory disease of poultry caused by orthomyxoviridae type A Influenza virus.The disease has the characteristics of acute onset,rapid spread and wide epidemic scope,which has caused huge economic losses to poultry breeding industry in China.Avian Influenza virus（AIV）is divided into many subtypes according to the antigenic characteristics of hemagglutinin（HA）and neuraminidase（NA）,including 18 HA subtypes（H1-H18）and 11 NA subtypes（N1-N11）.H9N2 AIV is endemic in all kinds of poultry in all countries of the world,and can infect mammals and even humans.The prevalence of H9N2 in China is very complex.In order to investigate the prevalence and pathogenicity of H9N2 in Shandong Province,274 suspected cases were collected from 2020 to 2021.Autopsy observation,histopathological diagnosis and etiological diagnosis of infected chickens were carried out,and the virus was isolated and purified.The whole genome of the eleven isolated strains was amplified and sequenced,and homology,genetic evolution,key amino acid sites and glycosylation sites of the isolated strains were analyzed.Four H9N2 AIV strains were selected to infect SPF mice.Some of the mice were killed after3 days,and the tissues and organs of the mice were collected for the determination of virus titer and the preparation of pathological tissue sections.The remaining mice were observed for 14 days and their mental state and body weight were recorded.In addition,serum samples from layer farms in some areas of Shandong province were collected for H9N2 AIV antibody detection,aiming to understand the immunization status of H9N2 AIV vaccine in Shandong province and provide reference for vaccine development and scientific immunization program.The results showed that 73 H9N2 positive samples were collected from 274 samples,with a positive rate of 26.64%.The detection rate of the virus was higher in winter.Eleven H9N2 AIV strains were isolated by inoculation in allantoic cavity of chicken embryo.The homology comparison between the isolates and the control strains showed that the isolates had the highest homology with the G57 genotype reference strain.According to genetic evolution analysis,the composition of 8 gene fragments of all strains was consistent with G57 genotype,so all 11 isolated strains belonged to G57 genotype.By analyzing the key amino acid sites of the isolated strains,11 H9N2 AIV strains were found to have mutations,such as T163,N191,V198 and L234 of HA protein,V588 of PB2 protein,V368 of PB1 protein and R356 of PA protein.These mutations greatly improved the adaptability of the viruses to mammals.All isolated strains have only a single alkaline amino acid on the HA cleavage site,which belongs to the molecular characteristics of low pathogenic avian influenza（LPAI）virus,so the isolated strains are LPAIV.In this study,31 N amantadine resistance mutations were found in all H9N2 AIV isolates,but no mutations in Neuraminidase inhibitors（NAIs）resistance were found,indicating that the isolates were resistant to M2 protein inhibitors but sensitive to NAIs.The results of titer detection of infected mice showed that most H9N2 AIV could replicate in the turbinate and lung of mice,and the replication efficiency of 3 H9N2 AIV strains in the turbinate was better than that in the lung.Suggesting that most strains are more capable of replicating in the upper respiratory tract.A total of 5152 serum samples were collected from 2020 to 2021.The average antibody titer of H9N2 AIV was 8.71 log2,the dispersion was 23.76%,the qualified antibody number was 4725,and the qualified antibody rate was 91.71%.The results showed that H9N2 AIV laying hens in Shandong province had higher antibody level and could resist the infection of H9N2 AIV well.