云南省芒市采集蠓中辛德毕斯病毒的分离与鉴定  

Isolation and identification of Sindbis virus from midges collected in Mangshi,Yunnan province,China

作　　者：何于雯 李楠[1] 孟锦昕 王静林[1] HE Yu-wen;LI Nan;MENG Jin-xin;WANG Jing-lin(Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory,Yunnan Animal Science and Veterinary Institute,Kunming,Yunnan 650224,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,云南昆明650224

出　　处：《中国媒介生物学及控制杂志》2021年第4期498-502,共5页Chinese Journal of Vector Biology and Control

基　　金：国家自然科学基金(31660714);云南省科技厅重点基金(2019FA015);云南省程功专家工作站(202005AF150034);云南省科技人才和平台计划项目(2018HB046);公益性行业(农业)科研专项资助项目(201303035);云南省畜牧兽医科学院基础研究项目(2019RW006,2019RW007,2019RW009)。

摘　　要：目的了解云南省德宏傣族景颇族自治州(德宏州)芒市蠓携带虫媒病毒情况,为虫媒病毒的研究和防控提供科学依据。方法2013年7月在德宏州芒市郊区牛和羊的养殖场中采用灯诱方法收集蠓标本。对采集到的蠓进行研磨离心,上清分别接种金黄地鼠肾细胞(BHK-21)和白纹伊蚊细胞(C6/36)进行病毒分离。阳性分离物分别采用甲病毒属、黄病毒属和布尼亚病毒属特异引物进行初步鉴定;采用辛德毕斯病毒(SINV)NS1基因特异性引物对阳性分离物进行反转录聚合酶链式反应(RT-PCR)扩增并进行测序及序列分析。结果共采集到4500余只蠓,分44批进行病毒分离,获得1株阳性分离物(编号:YN222)接种在BHK-21细胞24 h后迅速产生明显细胞病变,接种在C6/36细胞72 h后产生明显的细胞病变。甲病毒属引物RT-PCR扩增阳性,而黄病毒属和布尼亚病毒属引物扩增均为阴性。甲病毒属引物扩增阳性产物测序后BLAST结果显示YN222株分离物与MRE16株SINV核苷酸同源性最高为93.7%。采用MRE16株NS1基因特异性引物进行RT-PCR扩增结果为阳性,测序获得1605 nt序列。系统进化分析结果显示,蠓新分离病毒YN222与澳大利亚东方型SINV位于同一进化分支内,核苷酸和氨基酸同源性较高,核苷酸在93.7%~99.6%,氨基酸为99.4%。结论在云南省德宏州采集蠓中分离的病毒YN222为澳大利亚东方型SINV,这是首次在蠓中分离到SINV。Objective To investigate the status of arboviruses carried by midges in Mangshi,Dehong Dai and Jingpo autonomous prefecture(Dehong prefecture),Yunnan province,China,and to provide a scientific basis for the research and control on arboviruses.Methods The light-trap method was used to collect specimens of midges from cattle and sheep farms in suburbs of Mangshi,Dehong prefecture in July 2013.The collected Culicoides midges were ground and centrifuged,and the supernatants were inoculated with baby hamster kidney-21(BHK-21)and Aedes albopictus C6/36 cells separately for virus isolation.The specific primers for Alphavirus,Flavivirus,and Bunya virus were used to preliminarily identify the positive isolates,and the specific primers for Sindbis virus(SINV)NS1 gene were used to perform the reverse transcription polymerase chain reaction(RT-PCR),sequencing,and sequence analysis.Results A total of 4500 midges were collected,which were divided into 44 batches for virus isolation.One positive isolate(serial number:YN222)was obtained,which produced obvious cytopathic effect in 24 hours after inoculation of BHK-21 cells,and in 72 hours after inoculation of C6/36 cells.The RT-PCR amplification showed a positive result by using the primers for Alphavirus,whereas showed negative results by using those for Flavivirus and Bunya virus,suggesting that the isolate YN222 was from the genus Alphavirus.The BLAST sequence analysis after the amplification of positive products using the primers for Alphavirus showed that the isolate YN222 had a nucleotide homology of up to 93.7%with SINV MRE16 strain.The RT-PCR amplification showed a positive result by using the specific primers for NS1 gene of MRE16 strain,and a 1605-nt sequence was obtained via sequencing.The phylogenetic analysis showed that the newly isolated virus YN222 from midges was in the same evolutionary branch as the Oriental-Australian type of SINV,with a high homology in nucleotides(93.7%-99.6%)and amino acids(99.4%).Conclusion The virus YN222 isolated from midges collected in

关 键 词：辛德毕斯病毒 蠓 分离 鉴定 系统进化分析 

分 类 号：R373.3[医药卫生—病原生物学]