Molec?Lar Evolution And Pathogenicity Of H9N2 Avian Influenza Virus

H9N2 subtype avian influenza?AI?is widely prevalent in poultry in China.It can infect not only birds but also mammals,including humans.In order to clarify the molecular evolution and pathogenicity of H9N2 subtype avian influenza virus?AIV?in Shandong and its surrounding areas,37 strains of H9N2 AIV were isolated and identified from diseased chickens and ducks from 2016 to 2019.After virus purification,the whole genome sequence of each isolated virus was obtained by RT-PCR amplification and sequencing.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 of the 8 gene fragments from 37 H9N2 AIV strains belong to Eurasia lineage,but the molecular evolution of 8 gene fragments showed gene diversities.HA and NA genes of all viruses were from Y280-like lineage.The nucleotide homology among HA genes was 92.2%⁹9.9%,and the nucleotide homology among NA genes was 90.6%⁹9.9%.Both M and NS genes were derived from G1-like lineage.PA,NP,and PB1 genes were from F98-like lineage.PB2 gene came from G9-like lineage.The receptor binding sites 149,198,234and 235 of HA protein had the most variations.All of the 37 isolates had a 234L residue that equal to the 226 residue of H3 numbering and a 235M residue.The potential glycosylation site at residue 218 of HA protein was lost in 32 isolates,and all isolates contained an additional potential glycosylation sites at residue 313.Only 3 isolates had a potential glycosylation site at position 399 of NA protein,while 13 isolates possessed a new potential glycosylation site at residue 264.All these H9N2 viruses contained a three-amino-acid deletion at position 62 to 64 in the NA stalk region.For PB2 protein,six isolates were mutant at residue 627 and one isolate mutant at residue 714.Twelve H9N2 AIV strains with evolution distance of HA gene and significant difference in nucleotide homology were selected for antigenicity analysis.The results showed that there were differences in cross HI and R values between H9N2 AIV strains and partial strains showed significant differences in their antigenicity.The results demonstrated that the H9N2 AIVs prevalent in Shandong province and the surrounding areas in the past four years were evolved from different mode,and the virus antigenicity had changed.Some important amino acid sites were mutated,which enabled the virus a greater potential to enhance the capacity of infecting mammals.Therefore,it is necessary to strengthen the isolation and identification of H9N2 AIV and closely monitor its genetic variation.Based on the phylogenetic analysis and nucleotide homology of HA genes,the biological activity of representative strains with far distance from HA gene phylogenetic tree and significant difference in nucleotide homology were determined.The infectivity and pathogenicity of isolates from different years were evaluated in embryonated chicken eggs,Madin-Darby canine kidney?MDCK?cells,SPF chickens and BALB/c mice.The results showed that H9N2 AIV representative strains isolated between 2016 to 2019 had good adaptability in SPF chicken embryos,with EID₅₀ values ranging from 10⁶ to 10^9.25.The TCID₅₀ values measured on MDCK cells were between 10⁰.75.75 and 10⁹,which showed significant difference.All representative strains could replicate in chickens and shed virus,but the virus titers were low.Of the 21 representative strains,19 viruses could replicate in the lungs of mice without adaptation,and most strains had relative high virus titer and caused obvious pathological damage and weight loss.The results demonstrated that the replication capacity of H9N2 AIV isolates in vitro and in vivo of mammals were different,and the infection capacity in mouse showed an upward trend relative to that of chickens in the past four years.Therefore,continuous surveillance of the transboundary infectious capacity of prevalent H9N2 virus in poultry should be strengthen to prevent any devastating human influenza pandemics.In order to explore the molecular mechanism of enhanced ability of AIV to infect mammals caused by HA gene mutations,this study screened key HA amino acid mutation sites that might enhance infection capacity of AIV in mammals through the above continuous epidemiological investigation,combined with bioinformatics technology.Mutant viruses were further rescued by plasmid-based reverse genetics of H9N2 AIV and side-directed mutagenesis.Biological characteristics of mutant viruses were determined and the differential HA-host interaction proteins were identified.Collectively,nine mutant viruses were successfully rescued in this study.The TCID₅₀ values of mutants CK/SD/903?N313Q?,CK/SD/903?T198I?,CK/SD/903?M235Q?and CK/SD/903?M235R?were slightly higher than that of the parental virus.The loss of a glycosylation site at residue 218 of HA protein reduced the infectivity of H9N2 AIV in mammalian cells.The HA proteins of wild strain CK/SD/903 and mutant CK/SD/903?N218Q?shared 27 host interaction proteins,which mainly participated in signal pathways such as antigene processing and presentation,Ribosome,Spliceosome,etc.The differential interaction proteins participated in the pathways of antigen processing and presentation,protein processing in endoplassic reticulum,pathogenic Escherichia coli infection,etc.The results demonstrated that glycosylation at position 218 and mutation of some amino acids at residues Thr198 and Met235 of HA protein could enhance the proliferation of H9N2AIV on mammalian cells.Glycosylation at position 218 of HA protein might enhance the infectivity of the virus in mammals by influencing pathogenic infection,antigen presentation,virus protein synthesis and other processes.The results of the study provided a new perspective for studying the adaptation and pathogenicity of H9N2 AIV in mammals,and laid a foundation for further exploring the mechanism of transboundary infection capacity of H9N2 AIV.