2019—2020年我国6省市人副流感病毒3型全基因组序列特征分析

Analysis of genomic characteristics of human parainfluenza virus 3 in six provinces and cities of China,2019-2020

目的本研究旨在分析2019—2020年期间我国6个省市(北京、河南、吉林、安徽、甘肃和山东)流行的人副流感病毒3型(human parainfluenza virus 3,HPIV3)的全基因组序列特征,以揭示其基因组的遗传变异特点和分子进化规律。方法根据基因型别、遗传差异和时空分布等原则,从6个省市筛选出12株HPIV3流行代表株(其中7株为C3a型、2株为C3b型、3株为C3f型)。采用巢式RT-PCR方法进行分段重叠扩增并获取其全基因组序列。随后,构建全球HPIV3代表株的全基因组序列数据库,使用生物信息学软件开展分析。结果研究发现,这12株HPIV3代表株的全基因组序列长度在15227 bp~15370 bp之间,G+C含量为35.1%~35.3%,核苷酸一致性为97.6%~99.6%,与原型株(GenBank登录号:NC_001796.2)的核苷酸一致性为94.2%~94.5%。分析我国和全球可获取的HPIV3全基因组序列显示,基因组变异方式主要为点突变,未发现碱基缺失和基因重组现象。仅在本研究的一株吉林省代表株(CHN/Jilin036/2019/C3b)的F基因3′UTR区域发现ATTAAA碱基插入,其病原学意义有待进一步研究。对基因组编码蛋白的氨基酸比对结果显示,我国和全球广泛流行的C3a分支HPIV3在N蛋白、P蛋白和L蛋白上存在分支特异性变异位点,而我国流行的部分C3a毒株在L蛋白上还存在一个独特的变异位点(N216S),尚未发现我国C3b和C3f分支毒株存在特异性变异位点。基于全基因组的进化分析显示,全球HPIV3流行株的最近共同祖先株形成时间(the time to the most recent common ancestor,tMRCA)可追溯至1927年(95%HPD:1901—1945),平均分子进化速率为5.29×10^(-4)替换/位点/年,而我国HPIV3流行株的平均分子进化速率为5.24×10^(-4)替换/位点/年。此外,HPIV3编码的各个基因均受到负向选择压力,其中P基因、HN基因和F基因的核苷酸变异最为显著,且其分子进化速率高于其他基因。结论本研究期间6个省市流行的HPIV3病毒株全基因组进化相对保守,点突变是驱动HPIV3基因组进化的主要方式。

Objective This study comprehensively analyzed the genomic characterizations of human parainfluenza virus type 3(HPIV3)strains circulating in six provinces and cities of China(Beijing,Henan,Jilin,Anhui,Gansu,and Shandong)during the period of 2019-2020.The aim was to elucidate the intricate genetic variations and molecular evolutionary trends within the HPIV3 genome.Methods Based on genotypic differentiation,genetic divergence,and spatial and temporal distribution,12 representative HPIV3 strains(including 7 of C3a subtype,2 of C3b subtype and 3 of C3f subtype)were selected from the aforementioned provinces,and the complete genome sequence was successfully obtained by overlapping amplification of fragments using nested RT-PCR.Subsequently,a complete genome database of global representative HPIV3 strains was constructed and analyzed using bioinformatics tools.Results The length of complete genome of the 12 HPIV3 strains in the present study varied between 15227 bp and 15370 bp,the G+C content ranged from 35.1%to 35.3%and the nucleotide identity intermediated from 97.6%to 99.6%.Compared with the prototype strain(GenBank accession number:NC_001796.2),the nucleotide identity of 12 HPIV3 strains ranged from 94.2%to 94.5%.Analysis of the complete genome of HPIV3 available in China and globally showed that the genomic variation of HPIV3 was mainly shaped by substitution mutations,and no base deletions or gene recombination were observed.Only a six-base insertion(ATTAAA)was found in the F gene’s 3′UTR region of a representative strain originating from Jilin province(CHN/Jilin036/2019/C3b)in this study,and its potential pathogenic significance needs to be further investigated.Amino acid analysis of the encoded proteins revealed that the C3a lineage of HPIV3,widely prevalent both in China and worldwide,exhibits lineage-specific mutation sites in the N,P and L proteins.Furthermore,within the Chinese prevalent C3a strains,a distinctive mutation site(N216S)in L protein was also identified.Notably,specific variant sites have not been found in Chinese C3b and C3f branch strains.Based on the complete genome,the comprehensive evolutionary analysis showed that the time to the most recent common ancestor(tMRCA)of global HPIV3 strains was estimated to 1927(95%HPD:1901-1945),with an average molecular evolutionary rate of 5.29×10^(-4) substitutions/site/year,while the average molecular evolutionary rate of HPIV3 strains in China is 5.24×10^(-4) substitutions/site/year.In addition,each gene of HPIV3 was subjected to negative selection pressure,with the P,HN and F genes showing the most significant nucleotide variation and higher rates of molecular evolution than the other genes.Conclusions This study reveals that the complete genome of HPIV3 strains circulating in six provinces and cities of China tend to evolve conservatively.Moreover,substitution emerge as the main driving force for molecular evolution of HPIV3.