新一代全基因组测序揭示2014~2018年云南省临沧市H3N2亚型流感病毒的遗传突变机制

Next-Generation Sequencing Reveals the Mechanism of Genetic Mutation of the Influenza A (H3N2) Virus in Lincang,China,from 2014 to 2018

季节性H3N2亚型流感病毒进化速率快,极易发生抗原漂移,引起区域流行且致死率较高。为阐明云南省临沧市2014~2018年H3N2亚型流感病毒的遗传突变机制,选取临沧市2014~2018年H3N2亚型流感病毒29株,通过全基因组核酸提取、扩增、文库构建以及Illumina MiSeq测序获得基因组全序,并利用生物信息学软件比对基因组序列同源性、构建系统发育树以及解析氨基酸的变异规律。结果显示,29株H3N2亚型流感病毒的8个基因片段的基因进化树拓扑结构基本一致,17株毒株(2014年6株,2015年6株,2016年1株,2018年4株)隶属3C.3a进化支系,11株毒株(2015年1株,2016年1株,2017年7株,2018年2株)隶属3C.2a1进化分支,且这11株毒株出现3C.2a1进化支系特有的氨基酸突变(N121K、N171K、I406V和G484E),A/Yunnan-Linxiang/114/2018这一毒株在全基因组进化树上归属不同支系(例如:3C.3b、3C.2a、3C.2a1),提示临沧市2018年H3N2亚型流感病毒全基因组序列经历了快速进化。与相应年度疫苗株相比,所有毒株血凝素(Hemagglulinin,HA)蛋白重链HA1区抗原决定簇变异频率较高,T131K、T135K/N和A138S突变既位于抗原决定簇,又属HA蛋白受体结合位点(Receptor binding site,RBS),17株毒株HA1区潜在糖基化位点发生改变,且位于抗原决定簇上,15株毒株神经氨酸酶(Neuraminidase,NA)蛋白潜在糖基化位点发生突变,其中7株糖基化位点的突变发生在NA抗原决定簇上,这都会导致病毒抗原性发生改变,形成抗原漂移。本研究剖析了2014~2018年云南省临沧市H3N2亚型流感病毒起源和扩散的遗传突变机制,为云南省临沧市流感的科学防控提供了理论依据。

The seasonal influenza A(H3N2)virus appears a rapid evolutionary rate,and is prone to antigenic drift that causes regional pandemics with high mortality. We wished to elucidate the mechanism of genetic mutation of the influenza A(H3N2) virus in Lincang(Yunnan Province,China). Initially,29 influenza A(H3N2)virus strains were collected in Lincang,China,from 2014 to 2018. Whole-genome sequences were obtained by extraction,gene amplification,library construction and MiSeq ? sequencing(Illumina)of nucleic acids. Bioinformatics analysis was done using homologous alignments of whole-genome sequences,constructing phylogenetic trees and screening variants of amino acids. The whole-genome phylogenies from all of the eight gene segments for 29 influenza A(H3N2) virus strains supported approximately a consistent topological structure. Seventeen strains(six strains in 2014,six strains in 2015,one strain in 2016,and four strains in2018)were attached to the 3 C.3 a clade lineage,11 strains(one strain in 2015,one strain in 2016,seven strains in 2017,and two strains in 2018)were attached to the 3 C.2 a1 clade,and these 11 strains had 3 C.2 a1-specific amino acid mutations(N121 K,N171 K,I406 V and G484 E). Furthermore,one strain(A/Yunnan-Linxiang/114/2018)belonged to different branches in the genome-wide evolutionary tree(e.g.,3 c.3 b,3 c.2 a,3 c.2 a1),indicating that the whole-genome sequences of influenza A(H3N2) virus in Lincang,China,in 2018 had undergone rapid evolution. Compared with the corresponding annual vaccine strains,all strains had a higher mutation frequency in the antigen epitope of HA1,as well as T131 K,T135 K/N and A138 S mutations located in antigenic determinants and receptor binding sites(RBS). The potential glycosylation sites of 17 strains in the HA1 region were replaced and located in the epitope. Potential glycosylation sites of 15 strains in the NA protein experienced mutations,and the mutations from seven strains also occurred in the epitope of the NA protein. All of the mutations stated above would lead to antigenic variation of the virus,thereby forming an antigenic drift.Thus,the genetic-mutation mechanism of the origin and migration for influenza A(H3N2)virus from 2014 to2018 was confirmed. This research provides a theoretical basis for the scientific control and prevention of influenza in Lincang,China.