2013—2019年我国流行性出血病病毒血清1型毒株的分离与遗传特征分析  

Isolation and genetic characterization of epizootic hemorrhagic disease virus serotype 1 strains prevalent in China from 2013 to 2019

作　　者：李卓然 吴健敏[2] 朱建波[1] 王金萍[1] 吕敏娜 肖雷[1] 杨振兴[1] 廖德芳[1] 李华春[1] 杨恒[1] LI Zhuo-ran;WU Jian-min;ZHU Jian-bo;WANG Jin-ping;LYU Min-na;XIAO Lei;YANG Zhen-xing;LIAO De-fang;LI Hua-chun;YANG Heng(Yunnan Academy of Animal and Veterinary Science/Yunnan Key Laboratory of Tropical and Subtropical Animal Virus,Kunming 650224,China;Guangxi Veterinary Research Institute,Nanning 530001,China;Institute of Animal Health,Guangdong Academy of Agricultural Science/Guangdong Open Laboratory of Veterinary Public Health/Guangdong Provincial Key Laboratory of Animal Disease Prevention,Guangzhou 510640,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]广西兽医研究所,南宁530001 [3]广东省农业科学院动物卫生研究所/广东省兽医公共卫生公共实验室/广东省畜禽疫病防治研究重点实验室,广州510640

出　　处：《南方农业学报》2021年第8期2043-2052,共10页Journal of Southern Agriculture

基　　金：国家重点研发计划项目(2017YFC1200505,2016YFD0500908);农业部公益性行业(农业)科研专项(201303035);云南省中青年学术和技术带头人后备人才培养项目(2017HB055)。

摘　　要：【目的】掌握流行性出血病病毒(Epizootic haemorrhagic disease virus,EHDV)血清1型毒株(EHDV-1)在我国南方地区的流行情况及其遗传特征,为开展EHDV-1血清型特异性诊断、流行病学调查和致病性研究提供科学依据。【方法】2013—2019年通过在我国云南、广东和广西设立牛羊虫媒病毒监控点和哨兵牛,定期采集哨兵牛血液样本进行虫媒病毒分离;通过微量中和试验确定EHDV分离株的血清型,然后对EHDV分离株的部分基因节段(Seg-2、Seg-3和Seg-6)进行RT-PCR扩增、双向测序及序列分析,并利用BioEdit计算EHDV基因组各节段核苷酸序列及其推导氨基酸序列的相似性。【结果】2013—2019年从云南、广东及广西的虫媒病毒监控点共分离获得33株EHDV,其中7株为EHDV-1型毒株。分离获得的7株EHDV-1型毒株Seg-2、Seg-3和Seg-6核苷酸序列高度同源,其平均相似性分别为(97.65±1.51)%、(94.87±4.72)%和(97.61±1.41)%,对应的推导氨基酸序列相似性平均为(97.69±1.36)%、(99.49±0.32)%和(97.51±2.47)%。基于Seg-2、Seg-3和Seg-6核苷酸序列相似性构建的系统发育进化树均显示,不同EHDV-1型毒株可划分为东方(Eastern)和西方(Western)2种地域型,东方地域型毒株分离自中国、日本及澳大利亚,西方地域型毒株主要来源于美洲和非洲。在基于Seg-2核苷酸序列相似性构建的系统发育进化树中,分离获得的7株EHDV-1型毒株归属于西方地域型,且聚类形成一个相对独立的进化分支;在基于Seg-3核苷酸序列相似性构建的系统发育进化树中,分离获得的7株EHDV-1型毒株分属2种地域亚型(Eastern-1和Eastern-2),分别与日本EHDV-1型和EHDV-6型毒株具有最近的亲缘关系;在基于Seg-6核苷酸序列相似性构建的系统发育进化树中,分离获得的7株EHDV-1型毒株属于东方地域型,与日本EHDV-1型毒株具有较近的亲缘关系。【结论】EHDV-1型毒株已在我国南方地区广泛流行,其Seg-2和Seg-6序【Objective】Mastering the prevalence and genetic characteristics of epidemic hemorrhagic disease virus(EHDV)serotype 1(EHDV-1)strains in southern China provides basis for developing EHDV-1 serotype specific diagnosis methods,epidemiology investigation and pathogenicity research.【Method】Cattle and sheep arbovirus monitoring sites and sentinel cattle were set up in Yunnan,Guangdong and Guangxi from 2013 to 2019.Animal blood samples were collected regularly for arbovirus isolation.The serotypes of isolated EHDV strains were determined by neutralization test,and the genomic segments(Seg-2,Seg-3 and Seg-6)of the isolated EHDV strains were amplified using RT-PCR,bidirectionally sequenced and analyzed.BioEdit was used to calculate the identities of the nucleotide and deduced amino acid sequences.【Result】A total of 33 EHDV strains were isolated from sentinel animals between 2013 and 2019 in Yunnan,Guangdong and Guangxi,including 7 EHDV-1 strains.Sequence analysis showed that the Seg-2,Seg-3 and Seg-6 nucleotide sequences of EHDV-1 strains isolated from Yunnan,Guangdong and Guangxi sharedhigh similarty,with average sequence identities of(97.65±1.51)%,(94.87±4.72)%and(97.61±1.41)%,respectively.The average sequence identities of the corresponding deduced amino acid sequences were(97.69±1.36)%,(99.49±0.32)%and(97.51±2.47)%,respectively.The phylogenetic trees based on nucleotide sequence similarity of Seg-2,Seg-3 and Seg-6 indicated that EHDV-1 strains could be divided into Eastern and Western topotypes.The Eastern topotype strains were usual-ly isolated from China,Japan and Australia,while the Western topotype strains mainly came from America and Africa.The phylogenetic trees based on nucleotide sequence similarity of Seg-2 indicated that the 7 EHDV-1 strains belonged to the Western topotype and formed an independent evolutionary branch on the phylogenetic tree.The phylogenetic trees based on nucleotide sequence similarity of Seg-3 indicated that the 7 EHDV-1 strains belonged to Eastern-1 and Eastern-2 sub-t

关 键 词：流行性出血病病毒(EHDV) EHDV-1型 病毒分离 遗传特征 重配 

分 类 号：S852.65[农业科学—基础兽医学]