Study On The Pollution Status And Molecular Epidemic Characteristics Of AIV In The Environments Of The Live Poultry Market In Changsha

Objective: To investigate the pollution status of avian influenza virus(AIV)in different environments including air in a live poultry market(LPM)in Changsha city,and to understand the genetic evolution trend and molecular characteristics of these AIVs,so as to provide theoretical basis for the early warning of the risk of human infection caused by AIV in the environments of LPM and the prevention and control of AIV.Methods: From January to June 2021,different environmental samples of the LPM were collected for nucleic acid detection with AIV,and the distribution of AIV in different environments and at different times was analyzed.AIV was isolated and purified by the chicken embryo inoculation test,and their genome sequence information was obtained by high-throughput sequencing.The genetic evolution of AIV was analyzed based on homology comparison and phylogenetic tree construction,and the molecular characteristics of AIV were analyzed according to the mutations of key amino acid sites.Finally,homology modeling technology was used to predict the effect of related mutations on the protein structure of receptor binding site(RBS).Results:(1)In the first half of 2021,the detection rate of influenza A virus(Flu-A)in the environments of LPM in Changsha was as high as90.45%(161/178).Among different routine monitoring hemagglutinin(HA)subtypes of AIV,H9 had the highest detection rate(70.79%,126/178)(P<0.05),and the H5 combined with H9 had the highest detection rate(21.35%,38/178)among the mixed subtypes(P<0.05).Among different routine monitoring neuraminidase(NA)subtypes of AIV,N2 had the highest detection rate(60.67%,108/178)(P<0.05).(2)From January to June 2021,the positive rate of Flu-A fluctuated between 80% and 100% in each month,and the positive rate of H9 increased significantly in June.Multiple NA subtypes showed two peaks of the positive rate,and the transition time between the two peaks was about March to April.(3)Among different samples,poultry feces had the lowest degree of AIV pollution,which was the highest in cage swabs,cleaning poultry sewage,and the air samples near the poultry house.There was no significant difference in the degree of AIV pollution between the air samples near the poultry houses and the air near the depilator except H5subtype(P>0.05).(4)Twelve isolates of H9N2 subtype AIV had close genetic relationship with G57 genotype.Except for the MP gene,the other seven gene fragments obviously evolved into independent branches different from the reference strains of G57 genotype,and there was a certain genetic distance between these gene segments and the domestic H9N2 vaccine strains.(5)The HA protein cleavage site of the isolates had the motif of PSRSSR↓GL.The Q226 L mutation(H3 numbering)was found in RBS of all the isolates,and many mammalian adaptive mutations were found in the internal gene fragments.All isolates had some mutations in the antigenic sites of HA and NA proteins.Seven glycosylation sites of HA protein were predicted,which were missed the glycosylation site at position 218 and increased the glycosylation site at position 313 compared with some vaccine strains.Six glycosylation sites of NA protein were predicted,which changed more frequently at positions 264,368 and 402 compared with the vaccine strains.Neck deletion of NA protein was also found.All the isolates had the S31 N mutation in M2 protein.The N193 T,V223A and N224 H mutations in RBS of the isolates could cause some changes in the interaction forces that maintain the protein structure or the spatial conformation of the protein.Conclusion: In the first half of 2021,AIV was seriously polluted in the environments of this LPM,mainly with H9 and N2 subtypes.The change of building structure caused by market relocation may affect the distribution of AIV pollution.There was a high degree of AIV pollution in the air samples near the poultry houses and the air near the depilator,and its content may be related to whether the sampling time point was in the active trading period.The further isolated H9N2 subtype AIVs belonged to the widely prevalent G57 genotype,but it had a new trend of genetic evolution and mutation.Therefore,it is necessary to strengthen the monitoring of AIV in the environments including air of LPM and the development of new H9N2 vaccine.