蓝舌病毒HbC_3株S7基因5′非编码区序列分析

Sequence Analysis of S7 5′-NCR Gene of Bluetongue Virus-HbC_3 Strain

根据已发表的蓝舌病毒(bluetonguevirus,BTV)10型标准株S7基因设计合成一对与其5′ NCR(non codingregion)序列同源的引物,经反转录 聚合酶链反应(RT PCR)扩增出HbC3株和10型标准株长度分别为277bp和290bp的S7基因5′非编码区cDNA片段,以建立起dsRNA体外扩增系统.将扩增的BTV HbC3毒株的S7基因5′非编码区片段通过粘端连接克隆到pUCm T载体中,用PCR技术和限制性内切酶分析鉴定,表明获得重组质粒pUCm T BTV HbC3 S7.通过核苷酸序列分析方法分别将2个毒株的S7基因5′非编码区与呼肠孤病毒 3(reovirus 3)型比对,发现BTV HbC3株与呼肠孤病毒 3L2基因5′非编码区基因具有完全的同源性.将BTV HbC3毒株接种在不同细胞系如猴肾传代细胞(Vero)、人肝癌细胞(Hep 3B)和小鼠成纤维细胞(L929)等细胞株上,比较BTV HbC3在不同种系细胞上的增殖特征,并且用双向免疫扩散试验证实BTV HbC3和BTV 10型之间的血清学关系.结合本实验室的研究结果提示BTV HbC3株可能是蓝舌病毒的一个新的基因型.

Using primers complementary to the conserved sequence previously published of BTV genomic dsRNA segment,S7 5′-NCR of BTV-HbC_3 and BTV-10 were synthesized and amplified by RT-PCR method resulting in a 277 bp and 290 bp products. And the 277 bp product was then cloned into pUCm-T vector. The partial sequence of BTV-HbC_3 had a comparison with that of Reovirus-3 L2 5′-NCR revealing 100% nucleotide similarity,while it has no origin with BTV-10 in this part of sequence.Perhaps BTV-HbC_(3) and BTV-10 originated from two gene pools or some sequences of Reovirus-3 were inserted into BTV-HbC_(3) undergo genetic drift which contributed to diversification of individual gene segments of field strains of BTV. With the replicated and morphologic characteristics of BTV-HbC_(3) infecting different cells such as Vero,Hep-3B and L_(929) ,all the cells displayed a rounded, granular phenotype having the same CPE(characteristics of cytopathic effects) with that of BTV-10.Through agar gel immunodiffusion test,the cross-reactivities were detected between BTV-HbC_(3) and BTV-10 indicating that the common antigenic determinants on the VP7 proteins of BTV-HbC_(3) and BTV-10 shared some epitopes.All these findings showed that BTV-HbC_(3) was the prototype virus of the Orbivirus genus, probably a new genome type of bluetongue virus.