云南蚊虫中阿卡斑病毒的分离和鉴定  

Isolation and Identification of the Akabane Virus from Mosquitoes in Yunnan Province,China

作　　者：冯云[1] 何彪[2] 付士红[3] 杨卫红[1] 章域震[1] 涂长春[2] 梁国栋[3] 张海林[1] 

机构地区：[1]云南省地方病防治所,大理671000 [2]军事医学科学院军事兽医研究所,长春130062 [3]中国疾病预防控制中心病毒病预防控制所,北京102206

出　　处：《病毒学报》2015年第1期51-57,共7页Chinese Journal of Virology

基　　金：中美新发和再发传染病合作项目(U19-GH000004);NSFC-云南省联合资助基金/重点项目(No.U1036601)

摘　　要：为掌握云南省德宏州芒市和瑞丽市蚊媒病毒分布状况,2010年8月用诱蚊灯采集蚊虫17种2 149只,用金黄地鼠肾细胞(Baby hamster kidney cell,BHK-21)培养法分离病毒,阳性分离物用阿卡斑病毒(Akabane virus,AKV)S和M片段特异性引物的RT-PCR法鉴定。从采集蚊虫中分离到能引起BHK-21细胞病变和致乳小白鼠发病死亡的2株病毒,其中DHL10M110株分离自瑞丽市迷糊按蚊(Anopheles vagas),DHL10M117株分离自芒市三带喙库蚊(Culex tritaeniorhynchus)。RT-PCR扩增获得S片段的702bp序列和M片段的456bp序列,系统进化树分析表明DHL10M110和DHL10M117株与肯尼亚和澳大利亚AKV分离株进化关系较远;与日本、韩国和中国台湾的AKV分离株亲缘关系较近,但云南株形成一个独立的小分支。2株新分离病毒的S片段与中国台湾CY-77株的核苷酸(96.6%和96.7%)和氨基酸(99.6%和100%)同源性最高;M片段与日本Iriki株的核苷酸同源性最高(93.2%和93.6%),与中国台湾CY-77株氨基酸同源性最高(99.6%和100%);与肯尼亚MP496株核苷酸(69.7%和70.0%)和氨基酸(91.0%)同源性最低。本研究证实云南省德宏州存在AKV的流行,与亚洲流行株具有较近的亲缘关系。三带喙库蚊和迷糊按蚊可传播该病毒。To evaluate the prevalence of mosquito-borne viruses in Manshi and Ruili（Yunnan Province,China）,we collected 2 149mosquitoes（17species）in August 2010.Virus isolation was undertaken by the culture of baby hamster kidney cells（BHK-21cells）.Two virus-like isolates were obtained：DHL10M117was isolated from collected in Mangshi;DHL10M110was obtained fromAnopheles vagus collected in Ruili.Both isolates caused cytopathic effects,illness and death in suckling mice inoculated with these isolates via the intracerebral route.Two positive amplicons,702-bp from the S segment and 456-bp from the M segment,were obtained using reverse transcription-polymerase chain reaction using primers specific for the Akabane virus（AKV）.Phylogenetic analysis suggested that these two virus stains had a distant relationship with AKVs from Kenya and Australia,but were genetically close to those from Japan,South Korea,and Taiwan.However,they were separate from other Asian strains and grouped into a small branch.The highest nucleotide and amino-acid sequence identity of the S segment was found with the CY-77 strain from Taiwan（96.6%and 99.6%for DHL10M117 and 96.7%and 100%for DHL10M110,respectively）.Comparison of the M segment showed they shared the highest amino acid identity with CY-77（99.6% and100%,respectively）,whereas the highest nucleotide identity was found with the Iriki strain from Japan（99.6% and 100%,respectively）.Compared with the MP496 strain from Kenya,they displayed lower levels of sequence homology,at 69.7% and 70.0%for nucleotide sequences of the two loci,and 91.0%for amino acids.Our results identified that DHL10M117 and DHL10M110were strains of AKV,and provided molecular biological evidence for the existence of AKV in Yunnan Province.These AKV strains that are circulating in Yunnan Province share a close genetic relationship with strains from the rest of Asia.Culex tritaeniorhynchus and Anopheles vagus may serve as transmission vectors.

关 键 词：蚊虫 阿卡斑病毒 系统进化树 同源性分析 

分 类 号：R373.3[医药卫生—病原生物学]