血清15与21型蓝舌病病毒的分离与鉴定

摘要

[Objective] Bluetongue virus (BTV) is one of the most important arbovirus,which has many serotypes.The aim of this study is to characterize the epidemiology of BTV in Xuyi County of Jiangsu Province.[Method] A sentinel herd of 5 goats (BTV negative) was established in 2015.Blood samples were taken weekly from May to October and then monthly from November to April 2016 and tested for BTV antibody by C-ELISA.Serum samples (2 weeks,1 week and 0 week before sero-conversion time point) were tested by RT-PCR and the erythrocyte lysates from positive samples were inoculated into chicken embryo veins.Liver tissues of died chicken were harvested,homogenized in PBS and centrifuged.The supernatants were inoculated into C6/36 cells and passaged in BHK-21 three more times for virus isolation.The VP2 gene was detected by RT-PCR with a series of primers to identify the serotype of the isolates.[Result]The sero-conversion was observed from June in some animals and all animals sero-conversed in December.Five cell cultures showing cytopathic effect (CPE) were obtained finally.The VP7 gene was detected by RT-PCR and 1156 bp fragments were amplified from all five isolates.The results of VP2 gene detection of these five isolates showed that two isolates were determined to be BTV-15 and the other three were determined to be BTV-21.[Conclusion] This study provided useful information for the epidemiology of BTV in Jiangsu and laid foundation for prevention and control of BTV in the future.%[目的]蓝舌病病毒是一种重要的虫媒病毒,血清型较多,为了解近年来江苏省盱眙县蓝舌病病毒流行情况及血清型分别情况.[方法]本研究于2015-2016年在盱眙县桂五镇建立了5头蓝舌病血清学阴性山羊作为监控动物群.从2015年5-10月,每周采血1次,2015年11月至2016年4月,每月采血1次,用BTV C-ELISA试剂盒进行血清学监测.用RT-PCR检测转阳前2周、1周和转阳本周的血液样品,选择阳性样品处理后接种鸡胚,收获死亡鸡胚肝脏,用PBS悬浮捣碎的鸡胚肝脏,上清接种于C6/36细胞一代、BHK-21 3代进行病毒分离.阳性样品采用RT-PCR进行血清型鉴定.[结果]6月开始部分动物血清抗体转阳性,至12月,监控动物全部转为阳性.通过鸡胚、细胞传代培养共获得5个出现细胞病变的细胞培养物,经RT-PCR检测VP7基因均扩增出1156 bp片段.进一步通过RT-PCR检测VP2基因对5个分离毒株进行血清型鉴定.结果表明2株为BTV-15、3株为BTV-21.[结论]上述结果丰富了江苏省蓝舌病的流行病学资料,为蓝舌病的防控提供科学依据.