库蠓源蓝舌病病毒1型的分离和初步鉴定

Isolation and Initial Identification of Bluetongue Virus-1 from Culicoides Species in Yunnan Province,China

为监测云南边境地区虫媒库蠓蓝舌病病毒携带情况,本研究对2013年-2017年从云南6个口岸及周边地区采集到的约5 400只库蠓样本,分180组。采用荧光定量RT-PCR检测、鸡胚和细胞分离、目的基因克隆测序分析和间接免疫荧光试验等进行病毒分离与鉴定。结果显示:采集库蠓样本中有20组检出蓝舌病病毒核酸,检出率为11.11%(20/180);接种后有1份样本能导致鸡胚胚体充血出血和死亡以及BHK-21细胞呈现明显的细胞病变;RT-PCR能从感染细胞样本中扩增出蓝舌病病毒VP7基因特异性片段,且该片段序列与国外BTV-1毒株相应序列的相似性达95%~99%;间接免疫荧光试验显示分离病毒能与BTV-1抗体发生特异性结合。结果表明,云南边境地区库蠓携带有蓝舌病病毒,且为BTV-1,因此应加强对云南边境地区蓝舌病的预防与控制。

We wished to monitor carriage of the blue tongue virus(BTV)by Culicoides species(biting midges)in Yunnan Province,China. First,5400 samples of Culicoides species collected from six ports and surrounding areas of Yunnan Province from 2013 to 2017 were divided into 180 groups. The BTV was isolated and identified by:fluorescence quantitative polymerase chain reaction(RT-PCR);separation of chicken embryos and cells;sequencing of target genes;indirect immunofluorescence test. The nucleic acids of the BTV could be detected in20 groups of midges by real-time reverse transcription(RT -PCR),and the detection rate was 11.11%(20/180). After inoculation,one sample could lead to chicken-embryo congestion,hemorrhage and death,and BHK-21 cells showed obvious cytopathic changes. The specific VP7 fragment of the BTV could be amplified by RT-PCR from infected cell samples,and the sequence similarity between the fragment and the corresponding sequence of BTV-1 strain was 95% – 99%. The indirect immunofluorescence test showed that the virus could bind specifically to BTV-1 antibody. Our results showed that the BTV from Culicoides species in Yunnan Province was BTV-1.