云南省柯萨奇病毒A组6型全基因组特征分析

Characteristics of complete genome of Coxsackievirus A6 in Yunnan Province,China

目的分析2015年云南省4株柯萨奇病毒A组6型(Coxsackievirus A6,CV-A6)分离株的全基因组特征。方法取手足口病(hand,foot and mouth disease,HFMD)患儿粪便样本,制备悬液,接种至人横纹肌肉瘤细胞,CV-A6致细胞病变(CPE)后,收集病毒液,提取RNA,经RT-PCR分段扩增获得全基因组片段,并进行测序。采用Geneious 9.1.4和Mega 7.0、Simplot等生物学软件进行核苷酸和氨基酸序列比对、系统进化及重组分析,并构建系统进化树。结果4株CV-A6分离株之间核苷酸和氨基酸序列一致性分别为94.8%~97.9%和97.3%~98.4%,与近几年国内流行的CV-A6株核苷酸和氨基酸序列一致性分别为94.6%~97.9%和97.4%~99.1%;分离株在5′UTR、2A、2C、3A、3D、3′UTR区可能存在与毒力和临床表型相关的12个位点。结论4株云南分离株属于CV-A6血清型,为D3a基因亚型。

Objective To analyze the characteristics of complete genomes of four Coxsackievirus A6(CV-A6)strains isolated from Yunnan Province,China in 2015.Methods The stool samples of children with hand,foot and mouth disease(HFMD)were prepared into a suspension and inoculated into rhabdomyosarcoma(RD)cells.After cytopathic effect(CPE)appeared,the virus liquid was collected,from which the RNA was extracted for fractional amplification of complete genome fragments by RT-PCR and sequencing.The nucleotide and amino acid sequences were aligned to analyze gene evolution and recombination by using Geneious 9.1.4,Mega 7.0 and Simplot software,based on which a phylogenetic tree was constructed.Results The homologies of nucleotide and amino acid sequences among the four CVA6 isolates were 94.8%~97.9%and 97.3%~98.4%respectively.However,the homologies of nucleotide and amino acid sequences of the four isolates to those of CV-A6 strains epidemic in China in recent years were 94.6%~97.9%and 97.4%~99.1%respectively.There might be 12 sites associated with virulence and clinical phenotype in the 5′UTR,2A,2C,3A,3D,3′UTR regions of the four isolates.Conclusion The four strains isolated in Yunnan belonged to the D3a genotype of CV-A6.