WJBC株盖塔病毒基因组序列测定及与其他甲病毒的系统进化关系

Complete genome sequencing of one strain Getah virus and its phylogenetic relationship with other alphavirus

目的对WJBC株盖塔病毒(GETV)进行基因组序列测定,阐明其与已报道的GETV及其他甲病毒的关系。方法将GETV基因组编码区分12段分别进行RT-PCR扩增,将扩增产物直接进行测序,非编码区采用RACE法进行扩增,扩增产物纯化后连入pGEM-T easy载体,经转化DH5α感受态细胞,挑取阳性克隆鉴定后测序,应用DNAStar软件将测序结果拼接获得基因组序列。结果与结论 GETV基因组核苷酸共11 696 nt,编码3721个氨基酸,非结构基因编码2468个氨基酸,结构基因编码1253个氨基酸,结构基因和非结构基因之间有44 nt的连接区为非翻译区。病毒基因组5'端和3'端分别有78、411 nt的非编码区。序列同源性分析结果表明,此株病毒与M1株盖塔病毒同源性最高,两者核苷酸序列同源性为99.8%,氨基酸序列同源性为99.2%。

Objective To analyze the genome sequence of a Getah virus strain WJBC and compare its genome difference from other reported Getah virus and Alphavirus strains.Methods The coding sequences were amplified by 12 different RT-PCR reactions,and the noncoding region was amplified by RACE system and the sequenced cDNA fragments were analyzed by DNAStar software and spliced to a complete genome.Results and Conclusion The WJBC strain was 11 696 nt in length and had the common genome of Getah virus,encoding 3721 amino acids.The 5′ terminal two-thirds of RNA encoded four nonstructural proteins,nsP1,nsP2,nsP3 and nsP4.The 3′ terminal one-third of RNA encoded five structural proteins,C,E3,E2,6K and E1.A 44 nt untranslated region connected the nonstructural with the structural coding sequences.The noncoding region of genome was constituted by 78 nt of 5′ terminal and 411 nt of 3′ terminal.Phylogenetic analysis showed that the WJBC strain had the highest homology with M1 strain Getah virus： nucleotide sequence identity of 99.8% and an amino acid sequence identity of 99.2%.The results also showed that the WJBC strain had the highest homology with Sagiyama virus of Alphavirus： nucleotide sequence identity of 98.7% and an amino acid sequence identity of 97.8%.