柯萨奇病毒A组4型分离株全基因序列分析

Complete genome analysis of a coxsackievirus A4strain isolated from Yunnan Province,China

目的分析1株柯萨奇病毒A组4型(coxsackievirus A4,CV-A4)毒株的全基因组序列和系统进化特征。方法使用细胞培养法对肠道病毒阳性标本中的病毒进行分离。提取病毒RNA,通过RT-PCR法分段扩增全基因组,经测序、比对和组装后获得CV-A4分离株的全基因组序列。利用MEGA7.0软件分别基于全长VP1序列和全基因组序列构建系统发育树;使用Geneious Prime 2020.1.2软件对全基因组及编码区各区段的核苷酸和氨基酸序列同源性进行分析,并对编码区氨基酸变异情况进行比较分析。结果从人类横纹肌肉瘤(human rhabdomyosarcoma,RD)细胞上分离到1株CV-A4分离株,命名为R09220/YN/CHN/2009。基于全长VP1序列的系统进化分析显示,该分离株属于C2基因亚型。在全基因组核苷酸序列上与其同源性最高的是CV-A4毒株CVA4/SZ/CHN/09(核苷酸为95.4%,氨基酸为98.7%)。与C2基因亚型内16株CV-A4代表株的全序列核苷酸相似性为88.4%~95.4%,氨基酸序列相似性为97.5%~98.7%。通过比较C2基因亚型内的23株CV-A4分离株的全基因组氨基酸序列,R09220/YN/CHN/2009共存在23个氨基酸变异位点,其中10个为其独有的变异位点。结论R09220/YN/CHN/2009为肠道病毒CV-A4,属于C2基因亚型,该亚型在中国大陆流行的CV-A4病毒中占据优势地位。

Objective To analyze the genetic characteristics and phylogenetic evolution of the R09220/YN/CHN/2009 strain of Coxsackievirus A(CV-A)4isolated from Yunnan Province.Methods Cell culture was used to isolate the enterovirus strain from feces.Viral RNA was extracted from the supernatant when a cytopathic effect(CPE)was evident,and then a reserve transcription-polymerase chain reaction(RT-PCR)was used to amplify the complete genome in sections.The complete genome sequence was obtained after sequencing,comparison,and assembly.MEGA7.0was used to construct phylogenetic trees based on the full-length sequence of VP1and the complete genome.Geneious Prime 2020.1.2 was used to estimate the nucleotide and amino acid sequence homology of the whole genome and all of the segments of coding region and to analyze amino acid variation in R09220/YN/CHN/2009.Results The R09220/YN/CHN/2009 strain was only recovered from human rhabdomyosarcoma(RD)cells.A phylogenetic tree based on VP1indicated that R09220/YN/CHN/2009belongs to the C2sub-genotype.The topology of the phylogenetic tree based on the complete genome was very similar to that of VP1.The entire genome sequence of the R09220/YN/CHN/2009strain consisted of 7,421nucleotides.The nucleotide sequence of CVA4/SZ/CHN/09has the highest similarity to the nucleotide sequence of the complete genome of R09220/YN/CHN/2009(nucleotide sequence similarity:95.4%,amino acid sequence similarity:98.7%).The nucleotide sequence and amino acid sequence of the complete genome was 88.4%-95.4%similar to that of R09220/YN/CHN/2009and 97.5%-98.7%similar to that of the 16other C2reference strains.Comparison of the amino acid sequence of the whole genome of 23CV-A4isolates within the C2sub-genotype revealed 23amino acid variations in the R09220/YN/CHN/2009strain;10of them were unique and not found in any other C2strains.There were more amino acid variations in the VP1and 3Dregions of R09220/YN/CHN/2009.Conclusion R09220/YN/CHN/2009is a novel CV-A4strain and belongs to the C2sub-genotype,which is the predominant sub-genotype of CV-A4associated with hand,foot,and mouth disease in China's Mainland.