血清15与21型蓝舌病病毒的分离与鉴定

Isolation and Identification of Bluetongue Virus (BTV) Serotype 15 and 21 in Jiangsu Province

【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。

【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.