首次在云南省哨兵牛上分离出一种新型西藏环状病毒  

A Novel Serotype of Tibet Orbivirus Isolated from Sentinel Cattle in Yunnan Province,China

作　　者：李占鸿 杨振兴[1] 谢佳芮 高翔 胡忠燕 李卓然 廖德芳[1] 李华春[1] 杨恒[1,3] LI Zhanhong;YANG Zhenxing;XIE Jiarui;GAO Xiang;HU Zhongyan;LI Zhuoran;LIAO Defang;LI Huachun;YANG Heng(Yunnan Tropical and Subtropical Animal Virus Disease Laboratory,Yunnan Animal Science and Veterinary Institute,Kunming 650224,China;Animal Disease Control Center of Jinghong,Jinghong 666100,China;College of Agriculture and Life Sciences,Kunming University,Kunming 650214,China)

机构地区：[1]云南省畜牧兽医科学院云南省热带亚热带动物病毒病重点实验室,昆明650224 [2]云南省景洪市动物疫病预防控制中心,景洪655700 [3]昆明学院农学与生命科学学院,昆明650214

出　　处：《病毒学报》2021年第4期922-932,共11页Chinese Journal of Virology

基　　金：国家重点研发计划(项目号:2016YFD0500908),题目:牛羊虫媒病毒病诊断与检测新技术研究;国家重点研发计划(项目号:2017YFC1200505),题目:蓝舌病等虫媒性动物疫病现场快速检测与实验室鉴定技术研究;公益性行业(农业)科研专项(项目号:201303035),题目:重要牛羊虫媒病毒病防控关键技术研究与应用;云南省中青年学术和技术带头人后备人才培养项目(项目号:2017HB055)。

摘　　要：西藏环状病毒(Tibet orbivirus,TIBOV)为环状病毒属新成员,2009年首次在西藏墨脱县多斑按蚊中发现,目前该病毒对动物健康和公共卫生的危害尚不清楚,本文首次报道了牛体上一株新型TIBOV的分离。2019年在云南省景洪市设置哨兵牛3头,每周采血进行虫媒病毒的监测与病毒分离。监测期的第4周,其中一头牛采集的血液样本接种C6/36细胞盲传3代出现细胞病变,提取病毒核酸进行琼脂糖凝胶电泳,显示病毒基因组为10节段双链RNA,呈"3-3-3-1"的带型特征。电镜观察可见直径约70 nm,具有环状病毒属病毒典型形态特征的病毒粒子,将分离的病毒命名为V298/YNJH/2019。对决定环状病毒种属的基因节段及其编码蛋白(Seg-3/T2)序列分析显示,分离毒株的Seg-3/T2与其它TIBOV毒株的核酸(nt)与氨基酸序列(aa)相似度分别在79.6%~79.9%与96.3%~96.4%之间,其T2蛋白在系统发生树上与其它TIBOV聚为一簇;对决定病毒血清型的基因节段及其编码蛋白(Seg-2/OC1)的序列分析显示,分离毒株与其它TIBOV毒株Seg-2/OC1的序列相似度分别在42.3%~43.4%(nt)与29.3%~31.7%(aa)之间,其OC1蛋白形成独立于其它TIBOV毒株的进化分支,以上结果提示V298/YNJH/2019为一种新血清型TIBOV。通过实时荧光定量RT-PCR与血清中和试验,对病毒在牛上感染特征的回溯分析显示:在分离到病毒的前一周,动物血液中可检到低水平的病毒核酸,在分离到病毒的当周,血液中病毒核酸含量处于高峰,但两周后检测结果为阴性;动物在病毒感染后第2周开始产生中和抗体,在随后两周达到高峰,在感染后4个月仍保持较高水平。本研究首次报道了一种新型TIBOV在牛体上的分离,丰富了我国流行TIBOV多样性与感染特性的认知,为TIBOV的流行病学、致病性与诊断试剂的研究提供了基础。Tibet Orbivirus(TIBOV),a new member of the genus Orbivirus,was isolated for the first time from Anopheles maculatus collected in Motuo County,Tibet,China in 2009.Presently,little is known about the risk of the virus to animal health and public health safety.In this work,we reported a novel type of TIBOV isolated from cattle.To investigate prevalence of arbovirus,three cattle were set as sentinel animal in Jinghong City Yunnan Province in 2019.Blood samples were weekly collected from the cattle and inoculated on cells for virus isolation.One of the blood samples collected at Week four caused cytopathic effect on C6/36 after three passages.Viral RNA was extracted from infected cells and visualized using agarose gel electrophoresis which showed that the genome of the isolated virus was 10⁃segmented double⁃stranded RNA,showing"3⁃3⁃3⁃1"migration pattern.Through electron microscopy,the virus particles measured about 70 nm in diameter and shared typical morphological characteristics with Orbivirus.Analysis of Seg⁃3/T2 suggested the species of genus Orbivirus,indicating that V298/YNJH/2019 clustered together with other reported TIBOV strains in the phylogenetic tree.The identity of nucleotide(nt)and amino acid(aa)sequences were between 79.6%to 79.9%and 96.3%to 96.4%respectively,revealing that V298/YNJH/2019 is a member of TIBOV.Analysis of Seg⁃2/OC1 suggested the serotype of TIBOV,showing that the sequence identity of V298/YNJH/2019 with other TIBOV strains were between 42.3%to 43.4%(nt)and 29.3%to 31.7%(aa),respectively.Also,the OC1 of the V298/YNJH/2019 formed an independent branch on the phylogenetic tree,indicating that the V298/YNJH/2019 is a new serotype of TIBOV.Retrospective analysis of the neutralizing antibody level and viral nucleic acid in blood from the cattle infected by V298/YNJH/2019 showed that low level of viral nucleic acid was detected one week before the virus isolation and peaked in the week when V298/YNJH/2019 was isolated.After that,the viral nucleic acid in the blood droped rapidly

关 键 词：西藏环状病毒 新血清型 序列分析 感染特性 

分 类 号：S852.65[农业科学—基础兽医学]