2013-2020年潍坊市A(H1N1)pdm09流感病毒血凝素基因特征分析

Analysis on genetic characteristics of HA gene of influenza A(H1N1)pdm09 viruses in Weifang city,Shandong province from 2013 to 2020

目的分析2013-2020年山东省潍坊市A(H1N1)pdm09流感病毒血凝素(HA)基因遗传进化特征,了解潍坊市A(H1N1)pdm09亚型变异情况,为及早发现新变异株以及精准防控提供科学依据。方法收集2013-2020监测年度山东省潍坊市A(H1N1)pdm09流感病毒阳性样本,进行病毒分离,于阳性毒株中按年度共挑选18株流感毒株,采用二代测序方法获得HA基因序列信息后,使用RAxML软件,采用最大似然法(ML)构建系统进化树,使用BioEdit软件分析氨基酸位点变异情况,使用NetNGlyc 1.0 Server在线软件进行HA糖基化位点分析,使用datamonkey在线软件进行选择性压力分析,选用模型为单似然祖先计数(SLAC)模型、固定效应似然(FEL)模型。结果潍坊市18株A(H1N1)pdm09亚型分离株HA基因分属于6B、6B.1、6B.2等分支,大部分毒株(17/18)与对应年份疫苗株分属相同分支,且与对应年份疫苗株相比,HA基因序列的核苷酸同源性均>97.3%。氨基酸序列分析显示,HA蛋白有12~22个氨基酸突变不等,且均发生了位于抗原决定簇的突变,包括Sa区L161I、S162N、K163Q、S164T位,Sb区S185T、T185I位,Ca区H138Y、S203T位及Cb区S74R位,83.3%病毒发生了两个及以上抗原决定性突变;发现位于受体结合部位220环的Q223R突变及受体结合部位附近的S183P和I216T突变;自2016年开始潍坊分离株的糖基化位点改变较为频繁,包括11NSTD缺失、162NQSY增加,变异糖基化位点162NQTY增加及162NQTY缺失。选择性压力分析显示,dN/dS值分别为0.65与0.279,均未发现正向选择性压力位点。结论2013-2020年山东省潍坊市A(H1N1)pdm09流感病毒HA基因总体与当年疫苗株比较接近,但HA基因及其编码氨基酸仍存在持续变异,需加强监测。

Objective To analyze the genetic evolution characteristics of the HA gene of A(H1N1)pdm09 influenza viruses in Weifang city,Shandong province from 2013 to 2020,and to understand the mutation of the A(H1N1)pdm09 subtype,so as to provide scientific evidence for early detection of new variants and precise prevention and control.Methods From 2013 to 2020,positive samples of the A(H1N1)pdm09 influenza virus were collected in Weifang city,Shandong province,and virus isolation was subsequently performed.Over the years,18 strains of the virus were selected for next-generation sequencing to obtain their HA gene sequences.A phylogenetic tree was constructed using the Maximum Likelihood(ML)method with RAxML software,amino acid site variations were analyzed using BioEdit software,HA glycosylation sites were analyzed using the NetNGlyc 1.0 Server,and selective pressure analysis was performed using the DataMonkey online software with Single Likelihood Ancestor Count(SLAC)model and Fixed Effects Likelihood(FEL)model.Results The 18 A(H1N1)pdm09 isolates were categorized into subclades 6B,6B.1,and 6B.2.With nucleotide homology greater than 97.3%,most strains(17/18)belonged to the same subclade as the corresponding vaccine strains of each year.Amino acid sequence analysis revealed 12 to 22 mutations per strain,including mutations in antigenic determinants such as L161I,S162N,K163Q,S164T in the Sa region;S185T,T185I in the Sb region;H138Y,S203T in the Ca region;and S74R in the Cb region.Approximately 83.3%of the viruses had 2 or more antigenic determinant mutations.Mutations Q223R in the receptor-binding 220-loop and nearby S183P and I216T were also observed.Frequent changes in glycosylation sites began in 2016,including the loss of 11NSTD,addition of 162NQSY,and variations in 162NQTY.Selective pressure analysis showed dN/dS values of 0.65 and 0.279,with no positively selected sites identified.Conclusion From 2013 to 2020,the HA genes of A(H1N1)pdm09 influenza viruses in Weifang city were generally similar to those of the corresponding vaccine strains,but exhibited continuous variation at the nucleotide and amino acid levels,thus necessitating enhanced surveillance.