我国人副流感病毒3型HN基因的遗传变异及进化特征分析

Genetic variation and evolutionary characteristics of hemagglutinin-neuraminidase(HN)gene in human parainfluenza virus 3 in China

目的基于血凝素-神经氨酸酶蛋白(hemagglutinin-neuraminidase,HN)基因分析我国流行人副流感病毒3型(human parainfluenza virus 3,HPIV3)的基因变异规律和进化特征。方法采用多重荧光PCR方法对2007—2020年5个省市(北京、浙江、安徽、河南、湖南)急性呼吸道感染病例的咽拭子标本进行常见病原体核酸筛查,对HPIV3阳性标本进行HN基因序列测定,与GenBank数据库的国外和我国10个省的HPIV3流行代表株HN基因序列进行比对,应用多种生物信息学方法开展分子进化分析。结果本研究期间从5个省市共获得49株HPIV3的HN基因序列,核苷酸同源性在96.6%~100%之间,其中48株为C3基因亚型,1株为C5基因亚型。系统进化分析显示,我国12个省市在2003—2020年间存在C1、C3和C5基因亚型的毒株共流行,毒株间核苷酸和氨基酸同源性分别为95.4%~99.8%和97.9%~100%,其中C3是我国的优势流行基因亚型,分属于C3a、C3b、C3c、C3e和C3f进化分支,以C3f的传播范围和流行时间最为广泛。对C3基因亚型的进化分析显示,其最近共同祖先形成时间(tMRCA)可追溯到1990年,有效群体规模在2002—2010年间逐渐扩张而后趋于平稳;C3各进化分支HPIV3的HN基因进化速率在3.69×10^(-4)~5.82×10^(-4)替换/位点/年之间;C3基因亚型毒株HN蛋白共享N308、N351、N485和N523潜在N-糖基化位点,选择压力以负向选择为主。结论C3基因亚型是我国的本土优势HPIV3流行型别,形成了广泛传播和持续流行态势,并在流行过程中形成多个进化分支共同循环。

Objective To analyze the gene variation and evolutionary characteristics of human parainfluenza virus 3(HPIV3)circulating in China based on hemagglutinin-neuraminidase(HN)gene.Methods Multiple qPCR was used to screen nucleic acid of common pathogens in throat swabs from acute respiratory tract infection cases in five provinces(Beijing,Zhejiang,Anhui,Henan,Hunan)from 2007 to 2020.Subsequently,the HN gene sequence of HPIV3 positive samples was determined and compared with the HN gene sequence of HPIV3 strains from 10 provinces in China and abroad in the GenBank database.The molecular evolution analysis was carried out using a variety of bioinformatics method.Results A total of 49 strains of HPIV3 HN gene sequences were obtained from 5 provinces,with nucleotide homology ranging from 96.6%to 100%.Among them,48 strains were subtype C3 and 1 strain was subtype C5.Phylogenetic analysis showed that there were co-epidemics of C1,C3 and C5 strains in 12 provinces of China during 2003—2020,and the nucleotide and amino acid homology among the strains were 95.4%-99.8%and 97.9%-100%,respectively.Among them,C3 is the dominant subtype in China,which is divided into five evolutionary branches of C3a,C3b,C3c,C3e and C3f,and the C3f has the widest range and time of spread.The evolutionary analysis of the C3 subtype showed that the estimated time to the most recent common ancestor(tMRCA)of it dated back to 1990,and its effective population size tended to be stable after expansion from 2002 to 2010.The HN gene evolution rate of the evolutionary branches of C3 HPIV3 varied from 3.69×10^(-4) to 5.82×10^(-4) substitutions/sites/year;the HN protein of C3 subtype strains shared four potential N-glycosylation sites N308,N351,N485 and N523,and the selection pressure was mainly negative.Conclusions The C3 is an endemic dominant genotype,which has been widely spread and continuously circulating in China,and has formed different evolutionary clades during epidemic.