| H9N2 subtype avian influenza virus(AIV)was originally isolated from chicken in 1992 in Guangdong Province,China.Since then,the virus has spread nationwide and become endemic in poultry in many provinces of China,which is difficult to be completely eradicated.Over vicennial genetic evolution,the pathogenicity and transmissibility of the H9N2 subtype AIV have been increasing continuously,causing serious economic losses to the poultry industry.In addition,H9N2 subtype AIVs can break through barriers between species to infect humans directly.Moreover,H9N2 viruses also contribute to the ressortment of other subtypes of AIVs,such as the human-infected H5NX,H7N9 and H10N8.Therefore,H9N2 AIV have become a potential threat to public health.Although Genotype 57(G57)is still a dominant genotype of H9N2 subtype AIV at present,available evidence has demonstrated that during viral constant evolution,the adaptive substitutions in each gene segment,especially PB2,lead to the enhanced transmissibility and pathogenicity of H9N2 AIVs to mammals.Therefore,it's very necessary to continuous surveillance of H9N2 AIV and analysis of adaptive mutations in the PB2.In this study,we conducted the monthly epidemiological investigation on avian influenza viruses in live poultry markets(LPMs)and chicken farms in Eastern China from October 2018 to January 2020.H9N2 subtype AIV was characterized by phylogenetic analysis of the whole genome,and the effect of adptive amina acid substitutions in the PB2 gene on polymerase activity and the replication was also investigated.1.Epidemiological surveillence of H9N2 subtype AIV in LPMs in Eastern ChinaFrom October 2018 to January 2020,a total of 1,291 cloacal/laryngeal swabs samples were collected from live poultry markets(LPMs)in Eastern China,and 408 positive samples for AIV and Newcastle disease virus(NDV),accounted for 31.58%of total samples(408/1291),were identified through hemagglutination(HA)and hemagglutination inhibition(HI)tests.Among them,178 samples were positive for H9N2 subtype AIV.Meanwhile,105 strains of H9N2 subtype AIV were isolated and identified from clinical samples of commercial chicken farms.Hence,a total of 283 H9N2 isolates were obtained.Among the 408 positive samples isolated from LPMs,the positive rate of H9N2 subtype AIV was the highest(43.63%,178/408),followed by H5 subtype(31.86%,130/408),H3 subtype(15.68%,64/408),NDV subtype(7.35%,30/408),etc.The isolation rate of H9N2 in LPM during 2018.10-2020.1(13.08%-14.5%)was significantly higher than that of H9N2 in LPM during 2011.7-2016.9(0.21%-0.78%),and there was no significant difference from that during 2016.10-2018.9(8.49%-15.12%).The main host of H9N2 subtype AIV was still chicken in LPM(97.19%,173/178).Samples positive for H9N2 virus were mainly collected from Jiangsu,Shandong and Anhui provinces in Eastern China.The average separation rate of H9N2 subtype AIV in the hot summer was 11.98%,and there was no obvious seasonal difference.2.The genetic evolution of the H9N2 subtype AIV prevalent in poultry in Eastern ChinaIn this study,277 strains of H9N2 isolates were selected for sequencing of HA and NA gene,and 277 HA genes and 226 NA gene sequences were obtained.Based on the results of genetic evolution of HA and NA gene,55 representative isolates were selected for whole genome sequencing and phylogenetic analysis.Maximum Likelihood(ML)method of MEGA 7.0 was used to construct the phylogenetic tree.In our analyses,we observed that the HA and NA genes of all the viruses were evolved from the A/Duck/HongKong/Y280/97,and were distributed in clades 15 and clade 2,respectively.The PB2 and M genes of all the viruses distributed in clade 8 and clade 3,which were evolved from the A/Quail/HongKong/G1/97.The PB1,PA,NP and NS genes evolved from the A/Chicken/Shanghai/F98/98,and they were distributed in clades 5,7,6 and 7 in their respective phylogenetic trees.Therefore,all H9N2 subtype AIV strains belong to the S genotype(G57 genotype).However,a new branch,Group 3 of the HA gene,emerged,which involved only the H9N2 strains in the late 2018 and 2019.The HA protein of all isolates showed Gln(Q)to Leu(L)mutation at site 226,which affected the binding properties of viral receptors.Most isolates(61.5%,139/226)have 5 potential glycosylation sites in NA genes similar to WJ57/12,and the remaining isolates have 4(25.67%,58/226)or 6(12.83%,29/226)potential glycation sites,respectively.Other adaptive substitutions were observed in polymerase complex proteins,such as PB2 V1471(54/55),A184T(50/55),I292V(40/55),A588V(53/55),PB1 M317V(36/55),PAK356R(50/55),S409N(54/55),indicating that the virus showed a trend of increasing adaptability to mammalian hosts.3.The effect of adaptive mutation in PB2 on the replication of H9N2 subtype AIVThe sequences of the PB2 gene were collected from NCBI database and previous strudy in our laboratory.We compared the adptive substitutions in the PB2 gene between H and S genotypes,and found eight adaptive amino acid mutations(from genotype H to S),such as V147I?A184T?R353K?R389K?S590G?T598V?L648V and V661A.We selected representative strain AH320 of genotype S H9N2 virus as model virus,and introduced the combined mutations into the PB2 gene to assess the polymerase activity.The results showed that amino acid site mutation of I147V and/or T184A in PB2 resulted in the decrease in both the polymerase activity and viral replication.The combination mutation of I147V and T184A caused the most significant decrease in polymerase activity.In addtion,compared with the parent strain AH320,mutation of I147V,T184A,and I147V+T184A in PB2 gene could significantly reduce the copies of PB2 mRNA,vRNA,cRNA,the expression levels of PB2 protein and NP protein at 24h post infection.In conclution,we found that the adaptive mutations 147 and 184 in PB2 had a key affect on virus replication of the S genotype of H9N2 subtype AIV in mammalian cells.In brief,this study indicates that the S(G57)genotype H9N2 was still the dominant subtype of AIVs in live poultry markets in Eastern China during 2018-2020.In addition,HA proteins and polymerase complex of PB2,PB1 and PA proteins showed increasing number of mammalian adaptive markers.Moreover,147 and 184 mutation of PB2 gene influenced the replication level of the S genotype H9N2 virus in mammalian cells. |
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