云南省库蠓源蓝舌病病毒16型的分离鉴定  

Isolation and Identification of Bluetongue Virus Serotype 16 from Culicoides(Oecacta)reconditus in Yunnan Province

作　　者：付嘉佳 李富祥[1] 王金萍[1] 何于雯 谢佳芮 高华峰[1] 李小兵 FU Jiajia;LI Fuxiang;WANG Jinping;HE Yuwen;XIE Jiarui;GAO Huafeng;LI Xiaobing(Yunnan Tropical and Subtropical Animal Virus Disease Laboratory,Kunming 650224,China;College of Veterinary Medicine,Yunnan Agricultural University,Kunming 650500,China)

机构地区：[1]云南省热带亚热带动物病毒病重点实验室,云南昆明650224 [2]云南农业大学动物医学院,云南昆明650500

出　　处：《中国兽医杂志》2025年第4期37-43,共7页Chinese Journal of Veterinary Medicine

基　　金：云南地方羊种质资源开发平台建设及成果转化(202102AE090039)。

摘　　要：为了解云南省部分地区库蠓源蓝舌病病毒(BTV)流行情况,本试验于2023年7—10月分别从云南省昆明市宜良县和保山市龙陵县采集昆虫样品进行种属鉴定和虫媒病毒分离鉴定。基于昆虫的线粒体细胞色素氧化酶亚基Ⅰ(COⅠ)基因序列进行BLAST序列比对和种属鉴定。通过反转录聚合酶链式反应(RT-PCR)检测、病毒分离培养、间接免疫荧光试验和病毒毒价测定对所获得的虫媒病毒进行分离和鉴定,以及遗传进化分析。结果显示,采集的昆虫样品分别属于尖音库蚊、隐匿库蠓、拜氏铗蠓和蠓科。其中,采自云南省保山市龙陵县的样品RT-PCR检测结果为BTV阳性,经BHK-21细胞连续传代培养,得到1株BTV分离株;间接免疫荧光试验结果显示,分离株能与BTV-16标准阳性血清发生特异性反应,初步确定该分离株为BTV-16,将其命名为YNLL-BTV16-2023,其毒价为10-5.1/0.1 mL。遗传进化分析结果显示,分离株VP2基因序列与2株日本分离株基因序列(GenBank登录号:LC586239.1和AB686220.1)核苷酸同源性最高,分别为97.20%和96.87%,VP3基因序列与中国云南省分离株VP3基因序列(GenBank登录号:MG564322.1)核苷酸同源性为99.26%,VP5基因序列与日本分离株(GenBank登录号:AB686236.1)核苷酸同源性为97.77%,VP7基因序列与2株中国分离株(GenBank登录号:AF172831.1和KT002584.1)核苷酸同源性均为98.80%。结果表明,云南省保山市龙陵县边境地区存在BTV-16毒株,该血清型病毒可通过隐匿库蠓携带。本试验为云南省边境地区BTV及其传播媒介的分布提供参考。To investigate the prevalence of Culicoides-borne bluetongue virus(BTV)in certain areas of Yunnan Province,China,insect samples were collected from Yiliang County,Kunming City,and Longling County,Baoshan City between July and October 2023.Species identification and arbovirus isolation were performed.The species identification was conducted based on BLAST sequence alignment and mitochondrial cytochrome oxidase subunit I(COI)gene sequencing.The isolated arboviruses were analyzed through reverse transcription-polymerase chain reaction(RT-PCR),virus isolation and culture,indirect immunofluorescence assay(IFA),and virus titer determination,followed by genetic evolution analysis.The results showed that the collected insect samples included Culex pipiens,Culicoides reconditus,Forcipomyia bikanni,and Ceratopogonidae sp.Among them,samples collected from Longling County,Baoshan City,Yunnan Province,tested positive for BTV by RT-PCR.Following serial passage culture in BHK-21 cells,a BTV isolate was obtained.IFA confirmed that the isolate specifically reacted with BTV-16 standard positive serum,suggesting that the isolate belonged to BTV-16.This isolate was designated as YNLL-BTV16-2023,with a viral titer of 10-5.1/0.1 mL.Phylogenetic analysis revealed that the VP2 gene sequence of the isolate had the highest nucleotide homology with two Japanese isolates(GenBank accession number:LC586239.1 and AB686220.1),showing 97.20%and 96.87%similarity,respectively.The VP3 gene sequence exhibited 99.26%nucleotide homology with a Yunnan,China isolate(GenBank accession number:MG564322.1).The VP5 gene sequence had 97.77%nucleotide homology with a Japanese isolate(GenBank accession number:AB686236.1),while the VP7 gene sequence showed 98.80%nucleotide homology with two Chinese isolate(GenBank accession number:AF172831.1 and KT002584.1).These findings indicate the presence of BTV-16 strains in the border areas of Longling County,Baoshan City,Yunnan Province,with Culicoides reconditus serving as a potential vector for this serotype.This study

关 键 词：隐匿库蠓 蓝舌病病毒16型(BTV-16) 分离和鉴定 遗传进化分析 

分 类 号：S854.43[农业科学—临床兽医学]