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当前位置: 首页 > 文章 > 中国蓝舌病病毒血清5型毒株的分离与鉴定 中国兽医科学 2019 (1) 36-43
Position: Home > Articles > Isolation and identification of bluetongue virus serotype 5 strain in China Chinese Veterinary Science 2019 (1) 36-43

中国蓝舌病病毒血清5型毒株的分离与鉴定

作  者：
李占鸿;肖雷;孟锦昕;寇美玲;宋建领;廖德芳;高林;杨恒;李华春
单  位：
云南省畜牧兽医科学院热带亚热带动物病毒病重点实验室
关键词：
蓝舌病病毒;血清5型;分离;鉴定;序列分析;生物特性
摘  要：
为掌握我国云南省蓝舌病病毒(BTV)的流行情况,笔者在云南省师宗县设定哨兵动物,定期从哨兵动物上采集血液进行BTV分离。2012年笔者通过"鸡胚-C6/36细胞-BHK细胞"接种的方式从哨兵牛上分离出1株BTV(毒株号:V084/YN/2012),电镜观察显示,病毒粒子无囊膜,直径约80 nm,表面分布有纤突。血清中和试验表明,分离的病毒为蓝舌病病毒血清5型(BTV-5),使用抗BTV-5型的多克隆抗体为一抗进行间接免疫荧光染色,进一步证实分离毒株为BTV-5型。设计特异性引物对V084/YN/2012毒株的Segment 2 (Seg-2)与Segment 3 (Seg-3)的ORF区进行RT-PCR扩增与克隆测序,序列分析结果显示,V084/YN/2012与BTV-5型参考毒株(RSArrrr/05)的Seg-2核酸序列相似度为95.4%,在系统发育树上聚为一簇,属Seg-2基因E型;Seg-3在系统发生树上属Eastern topotype型,与中国BTV-4型YTS4毒株聚为一簇,核酸序列相似度高达97.5%。病毒蚀斑与增殖曲线测定试验表明,V084/YN/2012与实验室保存的BTV-5型参考毒株(RSArrrr/05)在BHK21细胞上可形成形态大小相近的病毒蚀斑,二者在BHK21细胞上的增殖特性也基本一致。本研究确认了BTV-5型V084/YN/2012毒株在我国的分离,掌握了病毒Seg-2与Seg-3基因的遗传特征以及病毒在BHK21细胞上的增殖特性。研究结果为进一步开展中国BTV-5型的基因组测序、流行病学调查与致病性研究奠定了基础。
译  名：
Isolation and identification of bluetongue virus serotype 5 strain in China
作  者：
LI Zhan-hong;XIAO Lei;MENG Jin-xin;KOU Mei-ling;SONG Jian-ling;LIAO De-fang;GAO Lin;YANG Heng;LI Hua-chun;Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory,Yunnan Animal Science and Veterinary Institute;
单  位：
LI Zhan-hong%XIAO Lei%MENG Jin-xin%KOU Mei-ling%SONG Jian-ling%LIAO De-fang%GAO Lin%YANG Heng%LI Hua-chun%Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory,Yunnan Animal Science and Veterinary Institute
关键词：
bluetongue virus;;serotype 5;;isolation;;identification;;sequence analysis;;biological characteristics
摘  要：
In order to study the prevalence of bluetongue virus(BTV) in Yunnan province of China,we set sentinel animals in Shizong county,Yunnan province and blood samples of the sentinel animals were regularly collected for the isolation of BTV.In 2012,a BTV strain(V084/YN/2012) was isolated from the sentinel cattle by inoculation with "chicken embryo-C6/36 cells-BHK cells".Virion was 80 nm in diameter,without envelope and distributed spikes on the surface under electron microscope.The serum neutralization test showed that V084/YN/2012 strain belonged to serotype of BTV-5.Using the polyclonal antibody against BTV-5 as the primary antibody,indirect immunofluorescence staining confirmed that the isolated strain belonged to serotype of BTV-5.Specific primers were designed to amplify the ORF regions of Segment 2(Seg-2) and Segment 3(Seg-3) of V084/YN/2012 and subsequently cloning and sequencing.The sequence analysis demonstrated that the Seg-2 of V084/YN/2012 strain showed 94.3% nucleotide identity to BTV-5 reference strain(RSArrrr/05),and clustered together in the phylogenetic tree,belonged to the Seg-2 nucleotype E.Seg-3 of V084/YN/2012 belonged to Eastern topotype and clustered with the Chinese BTV-4 strain(YTS4) in the phylogenetic tree,and the nucleotide sequence identity was as high as 97.5%.The results of plaque formation and proliferation curve assays indicated that V084/YN/2012 strain could form similar size plaques as the BTV-5 reference strain on the BHK-21 cells and the proliferation characteristics of the both virus strains was very close too.We confirmed the isolation of BTV-5 strain(V084/YN/2012) in China,elucidated the evolutionary characteristics of the virus Seg-2 and Seg-3 genes and the proliferation characteristics of the virus on BHK-21 cells.The results laid a foundation for further researches on the genome sequencing,epidemiological investigation and pathogenicity of BTV-5 in China.

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