我国蓝舌病病毒血清24型毒株的分离与遗传特征分析

Genetic characteristics of bluetongue virus serotype 24 strains isolated in China

掌握云南省蓝舌病病毒(BTV)的活动情况与流行毒株的遗传特征。2012—2015年,在云南省的师宗县、江城县与芒市分别设立3个监控点,进行BTV的分离。自监控动物采集的BTV核酸阳性血液通过"鸡胚-C6/36细胞-BHK细胞"接种的方式进行病毒分离;采用RT-PCR与中和试验进行分离病毒的血清型鉴定;设计特异性引物对分离病毒的Seg-2、Seg-3、Seg-7与Seg-6基因节段进行RT-PCR扩增与克隆测序。2012—2015年,从云南省的师宗县、江城县与芒市分别分离获得3株BTV-24型毒株。分离病毒的Seg-2、Seg-3、Seg-7与Seg-6序列系统发育分析显示:我国毒株的Seg-2与BTV-24型参考毒株聚为一簇,而Seg-6与BTV-10型毒株聚为一簇;我国毒株的Seg-3在系统发生树上划分为"东方型"地域型,Seg-7在系统发生树上形成一个独立于其他地域型的分支。本试验报道了我国BTV-24型毒株的分离与Seg-2、Seg-3、Seg-6与Seg-7序列特征,结果表明,我国BTV-24型毒株的Seg-6基因节段与BTV-10型毒株发生了基因重配;Seg-7具有独特的遗传特征,形成了一个新的Seg-7地域型,暂定为"Chinese topotype"。本试验为进一步开展BTV-24型的流行病学、感染特性与疫苗的研究奠定基础。

To study the prevalence of bluetongue virus(BTV)in Yunnan province and their genetic characteristics.Three BTV monitoring points were set at Shizong,Jiangcheng and Mangshi of Yunnan province.Virus isolation was carried out through the inoculating method of'chicken embryoC6/36cells-BHK cells'from BTV nucleic acid positive blood samples collected from the sentinel herds.The serotypes of the isolated BTV strains were identified by RT-PCR amplification and neutralization assay.Segment-2,-3,-6and-7of isolated viruses were amplified with specific primers,then cloned and sequenced.From 2012 to 2015,three strains of BTV-24 were isolated from Shizong,Jiangcheng and Mangshi in Yunnan province.Phylogenetic analysis of segment-2,-3,-6and-7of Chinese BTV-24 strains showed that segment 2of Chinese strains clustered with BTV-24 reference strain,however,the segment 6Chinese strains clustered with BTV-10 reference strain.Segment 3of Chinese BTV-24 strain was divided into'Eastern topotype',while segment 7formed an independent branch in the phylogenetic tree.This is the first report on isolation and sequence characterization of BTV-24 strain in China.The results showed that there existed reassortment between segment 6 of China BTV-24 strain and BTV-10 strain.Segment 7 of Chinese BTV-24 strains has unique genetic feature,forms a new topotype,tentatively designated as'Chinese topotype'.The results will lay foundation for further research on epidemiology,infection characteristics and vaccine of Chinese BTV-24.