摘要
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10 440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(x2=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.2alb.2进化分支,2022流感监测年(4-12月)属3C.2alb.2a.la.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.
Abstract
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 fluores-cent,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 amplifi-cation of whole genomes.The library was constructed and sequenced on a MiSeq sequencing system,and the sequencing data were ana-lyzed 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 10 440 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％;x2=41.26,P＜0.05)and amino acid variations(PB2 vs.NA:1.54％vs.7.45％;x2=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 influ-enza virus isolate was a 3C.2a cladein 2018(January to March),a 3C.2alb.2 clade between 2018 and 2020,and a 3C.2alb.2a.la.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.Conclu-sions 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 vi-rus are required to formulate the influenza control strategy.
NETL
NSTL
万方数据