柯萨奇病毒B组5型基因分型标准的研究

Study on genotyping criteria of coxsackievirus B5

目的依据肠道病毒分子分型的基本原则(基于VP1区全长核苷酸序列差异界值15%~25%),科学建立柯萨奇病毒B组5型(coxsackievirus B5,CVB5)的分子分型标准。方法本研究依托中国手足口病实验室网络,将来自中国大陆2001—2021年间分离出的69株CVB5采用逆转录-聚合酶链反应扩增全长VP1编码区,并进行核苷酸序列测定和分析;同时下载并筛选GenBank中截至2022年2月16日的742条CVB5全长VP1编码区核苷酸序列(849个核苷酸)。使用MEGA 7.0软件进行序列比对及构建基于最大似然法的基因进化树,自展值设定为1000,进行CVB5分子分型标准的研究。结果69株中国大陆CVB5分离株与CVB5原型株(Faulkner株)核苷酸和氨基酸的相似性分别为77.3%~81.6%和93.2%~95.3%。系统进化树显示,全球CVB5可分为4个不同分支,分支内核苷酸的平均差异范围在7.3%~13.4%,氨基酸的平均差异范围在1.9%~3.3%,分支间核苷酸的平均差异范围在17.0%~22.3%,氨基酸的平均差异范围为4.2%~4.9%。根据上述分子分型标准,将全球CVB5划分为A、B、C、D四个基因型。A基因型和C基因型CVB5可能在进化过程中已消失,不再流行,而B基因型和D基因型CVB5目前在全球呈现共流行趋势。中国大陆流行的CVB5主要为D基因型,少部分为B基因型。结论本研究建立了CVB5的分子分型的标准,并根据该标准将全球CVB5划分为4个基因型,对进一步了解CVB5的遗传变异变迁规律和进行分子溯源具有重要意义。

Objective To construct a scientific molecular typing criteria for coxsackievirus B5(CVB5)according to the basic principles of molecular typing for enteroviruses(based on the cut-off value of 15%-25%difference in the entire nucleotide sequence of VP1 region).Methods The entire VP1 coding regions of all 69 CVB5 strains isolated from mainland of China from 2001 to 2021 were amplified by reverse transcription-polymerase chain reaction using the Chinese Hand,Foot,and Mouth Disease(HFMD)Laboratory Network Platform.The nucleotide sequencing and analyses were also performed.The 742 complete sequences of VP1 coding region(849 nucleotides)of CVB5 in GenBank by the end of February 16,2022 were downloaded and filtered.The phylogenetic tree with maximum likelihood method for the study of CVB5 genotyping criteria was constructed using MEGA 7.0 software with the bootstrap value of 1000.Results Nucleotide and amino acids sequence similarity between 69 CVB5 isolates from the Chinese mainland and CVB5 prototype strain(Faulkner strain)ranged from 77.3%to 81.6%and 93.2%to 95.3%,respectively.The phylogenetic tree showed that CVB5 worldwide could be divided into 4 different branches.The average differences in nucleotides and amino acids within the branch were 7.3%-13.4%and 1.9%-3.3%,respectively.The average differences s in nucleotides and amino acids between branches were 17.0%-22.3%and 4.2%-4.9%,respectively.According to the above molecular genotyping criteria,CVB5 worldwide could be divided into four genotypes:A,B,C and D.Genotypes A and C might have disappeared during the evolution and were no longer prevalent.Genotypes B and D of CVB5 presented a co-epidemic trend worldwide.The predominant genotype of CVB5 in the Chinese mainland was genotype D with a small proportion of genotype B.Conclusions In this study,the criteria for molecular typing of CVB5 was established and the CVB5 strains worldwide were classified into four genotypes accordingly.This is of important value for further understanding of the genetic variation and transition of CVB5 as well as for molecular traceability.