2018—2022年无锡市H3N2流感病毒全基因组特征分析

Whole genome characteristics of H3N2 influenza virus in Wuxi City from 2018 to 2022

目的了解无锡市近年H3N2流感病毒的进化和变异特征。方法采用实时荧光定量RT-PCR方法,对流感样病例标本进行检测和分型,将H3N2流感病毒核酸阳性标本经细胞培养后,选取红细胞凝集试验(HA)≥1∶8的毒株扩增全基因组,构建文库,采用MiSeq测序仪上机测序,以NC_007366.1为参考株,使用CLC Genomics Workbench(Version 23)软件分析,采用MEGA 7.0软件构建系统进化树,NetNGlyc 1.0 Server软件预测N-糖基化位点。结果2018—2022年共监测流感样鼻咽拭子标本10440份,流感病毒核酸阳性率9.60%,H3N2型占28.44%(285株)。对19株H3N2型毒株进行全基因组测序分析,以HA基因核苷酸、氨基酸同源性最低,分别为97.43%~100.00%、96.37%~100.00%;M基因核苷酸、氨基酸同源性最高,分别为98.23%~100.00%、100.00%。不同基因核苷酸、氨基酸变异率差异均有统计学意义(χ^(2)=41.26、86.12,P值均<0.05),核苷酸变异率3.05%(M)~7.05%(HA),氨基酸变异率1.54%(PB2)~7.45%(NA)。基因进化距离以NS最小(0.000~0.028)、HA最大(0.000~0.065)。H3N2流感流行株2018流感监测年(1—3月)属3C.2a进化分支,2018—2020流感监测年属3C.2a1b.2进化分支,2022流感监测年(4—12月)属3C.2a1b.2a.1a.1进化分支。19株H3N2型毒株8个基因片段均有突变位点,以HA(51个)和NA基因(40个)较多,M基因较少(6个);5年均有特有突变位点,HA基因存在9~12个潜在N-糖基化位点,NA基因均为6个潜在N-糖基化位点。结论无锡地区H3N2流行株虽在不断进化,与疫苗株匹配性仍较好。应进一步加强监测,及时掌握流感病毒流行趋势,以制定有效的防控策略。

Objective To investigate the evolutionary and variation characteristics of H3N2 influenza virus in Wuxi City in recent years.Methods Nasopharyngeal swab specimens from influenza-like illness(ILI)cases were detected and genotyped using fluorescent,quantitative,real-time,reverse-transcription PCR(RT-qPCR)assay.Specimens tested positive for H3N2 influenza virus nuclear acid were cultured in cells,and virus isolates with 1:8 and higher titers of red blood cell haemagglutination assay were used for amplification of whole genomes.The library was constructed and sequenced on a MiSeq sequencing system,and the sequencing data were analyzed using the software CLC Genomics Workbench version 23 with NC_007366.1 as the reference strain.Phylogenetic tree was built using the software MEGA version 7.0,and the N-glycosylation sites were predicted using NetNGlyc 1.0 Server.Results A total of 10440 nasopharyngeal swab specimens were collected from ILI cases during the period from 2018 through 2022,and the proportion of positive influenza virus nuclear acid was 9.60%,with 285 H3N2 influenza virus isolates(28.44%).Whole-genome sequencing of 19 H3N2 influenza virus isolates showed the lowest nucleotide and amino acid homology in the HA gene(97.43%to 100.00%and 96.37%to 100.00%),and the highest nucleotide and amino acid homology in theM gene(98.23%to 100.00%and 100.00%).There were significant differences in the proportions of nucleotide(Mgene vs.HA gene:3.05%vs.7.05%;χ^(2)=41.26,P<0.05)and amino acid variations(PB2 vs.NA:1.54%vs.7.45%;χ^(2)=86.12,P<0.05).The minimal genetic evolutionary distance was seen in the NS gene(0.000 to 0.028),and the maximum distance was measured in the HA gene(0.000 to 0.065).The prevalent H3N2 influenza virus isolate was a 3C.2a cladein 2018(January to March),a 3C.2a1b.2 clade between 2018 and 2020,and a 3C.2a1b.2a.1a.1 clade in 2022(April to December).Mutational sites were found in 8 gene fragments of 19 H3N2 influenza virus isolates,with more sites in HA(51)and NA genes(40),and few sites in the M gene(6).In addition,specific mutational sites were found across the five-year study period,with 9 to 12 potential N-glycosylation sites in the HA gene and 6 potential N-glycosylation sites in the NA gene.Conclusions Continuous evolution of prevalent H3N2 influenza virus isolates is found in Wuxi City;however,the virus isolates match well with vaccine candidate isolates.Intensified influenza surveillance and understanding of trends in epidemiological features of influenza virus are required to formulate the influenza control strategy.