C组轮状病毒主要基因全片段扩增及武汉地方株Wu82的VP6和VP7编码基因序列分析

Amplification of the entire fragment of group C rotavirus genes and the sequence analysis on VP6 and VP7 genes in the field strain Wu-82 isolated in Wuhan area

目的建立C组轮状病毒VP6、VP7、VP4和NSP4基因全片段扩增方法;明确武汉地方株Wu82与其他毒株的系统发生关系。腹泻患者大便。方法PAGE筛查病毒;设计9对引物RT-PCR扩增VP6、VP7、VP4和NSP4基因;VP6、VP7序列及亲水位点分析。结果Wu82VP6和VP7基因核酸序列与其它人C组轮状病毒相比同源性分别为98.45%～97.12%和99.34%～94.83%。结论C组轮状病毒基因非常保守,进化缓慢。2001年武汉地方株Wu82与1995年武汉地方株208株相比,VP7和VP6抗原表位存在差异。

To establish a method to amplify the entire fragment of genes VP6 ,VP7 ,VP4 and NSP4 from group C rotavirus. In order to define the phylogenetic relations among the field strain Ww82 and other viral strains,4362 stool specimens from patients with diarrhea in all ages were collected during two periods of time,from Dec 2000 to Apt 2003 and from Nov 2003 to Feb 2007. The presence of rotaviruses in stool specimens was determined by detection of the dsRNA fragment of rotavirus by polyacrylamide gel electrophoresis,and the entire fragments of VP7,VP6, VP4 and nsp4 genes were amplified by RT-PCR with 9 pairs of specific primers. Nucleic acid sequence data and hydrophilicity analysis of the deduced amino acids were analyzed using the Gene Works software package and the MEGA 4 program. It was demonstrated that the VP6 and VP7 genes from Wu- 82 strain were quite conserved with few mutations,and the phylogenetic relation of the VP6 gene from Wu-82 strain was closer to the Nigerian strain Jajeri and Spanish strain BCN6, whereas the VP7 gene of this strain was closer to japanese strain E-93 and Columbia strain Javeriana. The sequence of the deduced amino acids of VP6 and VP7 of Wu-82 strain was distinct from that of the 208 strain isolated in 1995 at Wuhan area. Most of the different amino acids between Wu-82 and 208 strains was found to be located in the hydrophilicity regions. The homologies of nucleotide sequences of VP6 and VP7 genes with other group C rotaviruses were 98.45-97.12 % and 99.34-94.83 % respectively. From the observation of the present study,it is evident that the group C rotavirus genes are very conservative with slow process of evolution,but diversity of epitopes exists in Wu-82 and 208 strains.