云南省芒市蠓虫Tibet orbivirus和Manglie virus的分离及其共感染研究  

Isolation of Tibet Orbivirus and Manglie Virus from Midges and Its Coinfection Studies in Mangshi,Yunnan Province,China

作　　者：杨怡铃 何于雯 杨振兴[1] 陈易居 朱燕涛 殷建忠[2] 王静林[1] YANG Yiling;HE Yuwen;YANG Zhenxing;CHEN Yiju;ZHU Yantao;YIN Jianzhong;WANG Jinglin(Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory,Yunnan Animal Science and Veterinary Institute,Kunming 650224,China;Kunming Medical University,Kunming 650500,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]昆明医科大学,昆明650500

出　　处：《病毒学报》2023年第3期764-774,共11页Chinese Journal of Virology

基　　金：国家自然科学基金(项目号:32260896),题目:库蠓细胞系的建立及其在蠓传病毒研究中的应用;云南省科技厅重点基金(项目号:202201AS070062),题目:蚊虫特异性Negwotan病毒对乙型脑炎病毒传播影响的研究;云南省程功专家工作站(项目号:202005AF150034);云南省科技人才和平台计划项目(项目号:2018HB046),题目:云南省中青年学术和技术带头人后备人才;中央引导地方科技发展资金项目(项目号:202207AB110006),题目:云南省虫媒病毒病防控技术科技创新平台建设。

摘　　要：西藏环状病毒(Tibet orbivirus,TIBOV)是一种2009年新发现的环状病毒,它对家养动物具有潜在的危害。为了了解云南省西部边境地区芒市蠓虫中TIBOV流行情况,以及在TIBOV和Manglie virus(MaV)共感染情况下,MaV对TIBOV在细胞上增殖的影响。2018年7月我们在云南省德宏州芒市采集蠓虫标本,采用细胞进行病毒分离,获得2株阳性分离物,编号分别为MS18C217-8和MS18C217-9。MS18C217-8株阳性分离物接种C6/36细胞后72h出现细胞病变,细胞病变特点为圆缩、折光性变强和脱落等,而接种BHK-21细胞后168h没有出现明显细胞病变;MS18C217-9株阳性分离物接种BHK-21细胞后72h出现细胞变圆、脱落和破碎等细胞病变,接种C6/36细胞后168h没有出现明显细胞病变。分别采用虫媒病毒特异引物进行RT-PCR,MS18C217-9株阳性分离物TIBOV特异引物扩增阳性,而MS18C217-8株阳性分离物MaV特异引物扩增阳性。测序后,MS18C217-8获得1133个核苷酸序列,MS18C217-9获得1085个核苷酸序列。遗传进化分析结果显示,MS18C217-9株病毒与中国云南、广东、西藏等省、自治区和日本等地区分离的7株TIBOV位于同一进化分支,核苷酸同源性在93.1%~99.2%之间,提示MS18C217-9株病毒是TIBOV;而MS18C217-8株病毒与2011年和2018年云南分离的2株MaV位于同一进化分支,它们之间核苷酸同源性在96.9%~97.3%之间,提示MS18C217-8株病毒为MaV。TIBOV和MaV两种病毒在C6/36细胞上共感染研究结果显示,2种病毒共感染后TIBOV滴度低于TIBOV单独感染病毒滴度,提示MaV可能降低了共感染期间TIBOV复制。本研究首次在蠓虫中分离到MaV,并在同一地点采集的蠓虫中同时分离到了TIBOV和MaV,提示这两种病毒在当地的蠓虫中存在共流行。MaV在媒介中的感染可能会抑制TIBOV复制或传播。该研究结果不仅丰富了我国虫媒病毒种类资源,同时也为阻断动物虫媒病毒病的流行提供新的思路。Tibet orbivirus(TIBOV)was a new orbivirus discovered in 2009,which has potential harm to domestic animals.In order to understand the prevalence of TIBOV in Mangshi located in the western border of Yunnan Province,China and the effect of MaV on TIBOV's proliferation when TIBOV and MaV were coinfected in same cells.In July 2018,we collected midges in Mangshi,Dehong Prefecture,Yunnan Province,China.Cells were used for virus isolation,and two positive isolates were obtained,named MS18C217-8 and MS18C217-9.MS18C217-8 produced obvious cytopathic effect in Aedes albopictus cells(C6/36)72 hours after inoculation,the cytopathic features were roundness,refraction and shedding.There was no cytopathic effect in golden hamster kidney cells(BHK-21)up to 168 hours after inoculation.MS18C217-9 produced obvious cytopathic effect in BHK-21 cells 72 hours after inoculation,the cytopathic features were rounding,shedding and breaking.There was no cytopathic effect in C6/36 cells up to 168 hours after inoculation.RT-PCR was performed using arbovirus specific primers,MS18C217-9 showed a positive result with the primer of TIBOV VP7 and MS18C217-8 showed a positive result with the primer of MaV hypothetical.A 1133-nt sequence was obtained for MS18C217-8 and a 1085-nt sequence was obtained for MS18C217-9.The sequence analysis results showed that the MS18C217-9 strain was in the same evolutionary branch as the seven TIBOV strains isolated in Yunnan,Guangdong,Tibet and Japan,with a nucleotide homology of 93.1%-99.2%,suggesting that the MS18C217-9 is a TIBOV;the MS18C217-8 strain was in the same evolutionary branch as the two MaV strains which was isolated from Yunnan,China in 2011 and 2018,with a nucleotide homology of 96.9%to 97.3%,suggesting that the MS18C217-8 strain is a MaV.The results of coinfection with TIBOV and MaV showed that the TIBOV's titer was significantly lower than TIBOV alone,suggesting that the MaV may reduce TIBOV's replication during co-infection in C6/36 cells.In this study,MaV was first isolated in midges,both TIBOV a

关 键 词：西藏环状病毒 昆虫特异性病毒 蠓虫 分离鉴定 共感染 

分 类 号：S852.65[农业科学—基础兽医学]