浙江省嘉兴市2株与手足口病相关的柯萨奇病毒B3分离株的全基因组特征分析

Whole-genomic analysis of two coxsackievirus B3 isolates associated with hand,foot and mouth disease in Jiaxing,Zhejiang,China

目的分析浙江省嘉兴市2株与手足口病相关的柯萨奇病毒B3型(CVB3)毒株的全基因组序列特征,为手足口病的监测与防控提供科学依据。方法收集2021年浙江省嘉兴市辖区内7个区、县疾病预防控制中心上送至嘉兴市疾病预防控制中心的手足口病监测样本,经实时荧光定量反转录聚合酶链反应(real-time quantitative reverse transcription PCR,real-time qRT-PCR)检测后,选取肠道病毒核酸通用阳性的样本接种于人横纹肌肉瘤(human rhabdomyosarcoma,RD)细胞和人喉癌上皮(human larynx carcinoma,HEp-2)细胞进行病毒分离培养,对产生细胞病变效应(cytopathic effect,CPE)的阳性分离株进行高通量测序(Illumina NextSeq 550平台),鉴定出2株CVB3,并获得其全基因组序列。利用DNAstar软件对核苷酸序列及所编码氨基酸进行同源性分析;用MEGA6软件对VP1区进行基因分型,同时进行系统进化树的绘制;采用Simplot3.5.1软件进行重组分析。结果2株CVB3毒株基因组全长分别为7352 bp和7367 bp,其核苷酸和编码区氨基酸相似性分别为93.3%和99.5%。与CVB3原型株相比,2株毒株在编码区无碱基插入或缺失,在非编码区存在个别碱基的插入或缺失,与原型株的核苷酸相似性分别为78.9%和79.1%,编码区氨基酸相似性分别为96.0%和96.1%。VP1区系统进化树分析结果显示,本研究的2株CVB3属于E基因型,与2020年广东省分离株同属于国内新发现的分支(相似性95.09%~98.18%),与国外澳大利亚、日本分离株亲缘性最近。2株CVB3的全基因组及其P1、P2、P3区基因序列系统进化树和Simplot重组分析结果显示,2株CVB3在P2和P3非结构编码区的不同区段与不同血清型的肠道病毒有型间重组现象。结论嘉兴市的2株CVB3毒株属于国内新发现的E基因型,有重组现象,需关注CVB3的流行潜力并加强监测。

Objective To analyze the whole-genomic characteristics of two coxsackievirus B3(CVB3)strains associated with hand,foot and mouth disease(HFMD)in Jiaxing city,Zhejiang province,and to provide scientific basis for HFMD monitoring and control.Methods In 2021,HFMD surveillance specimens were collected by district-and county-level Centers for Disease Control and Prevention(CDC),and were submitted to Jiaxing municipal CDC.After real-time quantitative fluorescence reverse transcription PCR(real-time qRT-PCR),the positive specimens for enterovirus(EV)nucleic acids were inoculated with human rhabdomyosarcoma(RD)and human larynx carcinoma(HEp-2)cells for viral culture and isolation.The positive isolates that produced cytopathic effects(CPEs)were subjected to high-throughput sequencing in the Illumina NextSeq 550 platform.Two strains of CVB3 were identified,and their whole genome sequences were obtained.Homology analysis of the nucleotide sequences and the encoded amino acids were performed by DNAstar software;the VP1 region was genotyped with MEGA6 software,and the phylogenetic tree was drawn simultaneously;and recombination analysis was carried out using Simplot 3.5.1 software.Results The complete genomes of the two CVB3 strains were 7352 bp and 7367 bp in length,with nucleotide and coding amino acid similarities of 93.3% and 99.5%,respectively.Compared to the prototype CVB3 strain,the two isolates had no insertions or deletions in the coding region.However,occasional insertions or deletions in the non-coding region were found,with nucleotide similarities of 78.9% and 79.1% and coding amino acid similarities of 96.0% and 96.1%,respectively.Phylogenetic analysis of the VP1 region indicated that both CVB3 strains belonged to genotype E and clustered with isolates from Guangdong province in 2020,belonging to a newly identified domestic branch(similarity 95.09% to 98.18%),and had the closest genetic relatedness to isolates from Australia and Japan.The results of phylogenetic tree and Simplot recombination analyses of the whole genome of the two CVB3 strains and their gene sequences in the P1,P2,and P3 regions showed that there was intertypic recombination between the two strains of CVB3 in different segments of P2 and P3 non-structural protein regions and the different serotypes of EV.Conclusions Recombination exists in the two CVB3 strains in Jiaxing city which belong to the newly identified genotype E in China.It is essential to monitor CVB3 epidemic and strengthen the surveillance.