柯萨奇B组3型病毒中国分离株VP4、3D基因序列及系统发生树分析

Sequence and phylogenetic of VP4 and 3D gene of a Chinese isolate of coxsackievirus B3

目的研究柯萨奇B组3型病毒(CVB3)中国分离株结构蛋白VP4、非结构蛋白3D基因序列及变异性。方法在HeLa细胞中增殖病毒,用RT-PCR扩增目的基因片段,与pMD18-T载体连接,PCR初步鉴定后测序,进行序列同源性及系统发生分析。结果CVB3中国分离株VP4基因含207个碱基,编码69个氨基酸,与Nancy株氨基酸同源性为97.10%;3D基因含1386个碱基,编码462个氨基酸,与Nancy株氨基酸同源性为97.62%。在系统发生中,CVB3中国株VP4基因、3D基因均与Nancy株聚簇。结论CVB3中国分离株VP4和3D基因长度与Nancy株一致,进化上属同一分支。

Objective To investigate the sequence and variation of VP4 and 3 D gene of a coxsackievirus B3 Chinese isolate （CVB3-Chi）. Methods The virus was cultured in HeLa cells. Target gene fragments were amplified by RT-PCR and then cloned to plasmid pMD18-T vectors. PCR was used to identify whether each target fragment was connected to the plasmid, then positive clones were sequenced. Phylogenetie trees were then construeted for each region, using the sequence of CVB3-Chi, some other CVB3 strains and all other 36 HEV-B prototype strains of which full length of VP4 and/or 3D gene sequence data were obtained from GenBank. Sequence of poliovirus strain Sabin 1 （HEV-C） was used as an outgroup. Results The VP4 gene of CVB3-Chi comprise 207 nueleotides, encoding 69 amino acids; while the 3D gene comprise 1386 nucleotides, encoding 462 amino acids, sharing about 97.10% and 97.62% similarity with the amino acids of Nancy strain respectively. In both VP4 and 3D regions, analysis of phylogenetic trees showed that CVB3-Chi clustered together with Nancy and most of the other CVB3 strains. Incongruity was found between VP4 and 3 D trees according to the topological structure. Conclusion The length of VP4 and 3 D gene of CVB3-Chi is the same as that of Nancy strain. Moreover there may be a close evolutional relationship between both strains. The observation that incongruity between VP4 and 3 D trees support the hypotheses that recombination is a frequent event occurring in enterovirus during evolution.