肠道病毒71型广西株的全基因组序列分析

Analysis on the Completed Genome Sequencing of Enterovirus 71 Isolated in Guangxi

目的对2008年分离自广西壮族自治区柳州市手足口病死亡儿童的1株肠道病毒71型(Enterovirus Type71,EV71),进行了全基因组的序列测定,并对其分子变异和进化特点进行了分析。方法采用逆转录-聚合酶链反应(Reverse Transcription-Polymerase Chain Reaction,RT-PCR),分段扩增覆盖病毒全长序列的9个重叠片段(未包括多聚腺苷酸尾),并进行序列测定和分析。结果 EV71广西株基因组全长7405个核苷酸(nt),5'端和为3'端分别为742nt和84nt的非编码区,5'和3'非编码区间为一个6579nt长的开放阅读框,编码一个含2193个氨基酸的多聚蛋白,编码区内未见核苷酸的插入或缺失。在整个编码区,广西株与浙江株FJ158601的核苷酸及氨基酸序列同源性最高,为98.81%;在VP1编码区,与浙江株FJ158601、安徽阜阳株FJ439769核苷酸及氨基酸序列同源性最高,均为99.10%;在VP4编码区,与安徽阜阳株FJ439769的核苷酸及氨基酸序列同源性最高,为99.51%。结论首次完成广西EV71全基因组核苷酸序列的测定,经核苷酸序列同源性比对和进化树分析,证实EV71广西株属于C基因型中的C4基因亚型,与2008年国内各地区所爆发的EV71的亚型相吻合。遗传进化分析也发现,广西株与国内2008年各地区所分离的EV71毒株亲缘关系很近,提示可能源于同一种系。

Objectives To characterize the completed genome sequence and the sub-genotype of an enterovirus 71（EV71） strain which isolated from Liuzhou city of Guangxi.Methods Nine overlapping clones covering the whole viral genome（excluding the poly-A tail） were obtained by RT-PCR and sequenced,and their nucleotide and amino acid sequences were compared with other known EV71 strains.Results The genome of the EV71 Guangxi strain had 7405 nucleotides in length,and contained a 742-nt non-translated region（NTR） at the 5＇end and a 84-nt NTR at the 3＇end.The entire open reading frame contained 6579 nt,encoding a 2193-aa polyprotein.In the coding region,there was no nucleotide deletion or insertion,but some changes of amino acid were unique.The sequence alignments showed that the Guangxi strain shared the highest nucleotide and amino acid identities to the Zhejiang strain FJ158601 in the coding region,to the Zhejiang strain FJ158601 and the Fuyang strain FJ439769 in the VP1 region,and to the Fuyang strain FJ439769 in the VP4 region.Conclusion It is the first study of complete genome sequence of EV71 in Guangxi.The phylogenetic analysis based on the complete sequence and the VP1 and VP4 regions shows that the EV71 Guangxi strain belongs to C4 sub-genotype,which corresponds with the sub-genotype circulating in other areas of China during EV71 outbreaks in 2008.The phylogenetic analysis also reveals that the Guangxi strain is closely related to the strains isolated from other parts of China in 2008,indicats that the genetic linkages among those strains.