2015年从云南哨兵山羊中分离到阿卡斑病毒

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目的:了解云南省家畜阿卡斑病毒(akabane virus, AKAV)的流行情况。方法:2015年4月在云南省芒市设立5只山羊和10头黄牛作为哨兵动物进行家畜虫媒病毒监测,5~10月份每周采集动物血液,标本经实验室处理后接种BHK-21细胞进行病毒分离,对病毒分离物进行病毒基因(RT-PCR)扩增、核苷酸序测定及分析。结果:1只哨兵山羊血液样品接种BHK-21细胞48 h后产生明显细胞病变,取细胞培养物上清液脑内接种1日龄乳鼠,3 d后乳鼠开始发病死亡,经RT-PCR鉴定和S片段基因序列测定分析,分离毒株为AKAV(毒株编号16415)。16415毒株S片段基因全长856nt,编码N蛋白233aa和Ns蛋白91aa,遗传进化分析结果显示新分离16415毒株与国内外分离的AKAV位于同一进化分支内,核苷酸同源性在83.8%~97.7%之间;进一步分析发现16415株与广西2013年从竹鼠脑内分离到的AKAV亲缘关系最近,核苷酸和氨基酸同源性分别为97.7%、99.6%;与日本AKAV OBE-1毒株相比,16415株、广西竹鼠分离毒株和德宏迷糊按蚊分离毒株在N蛋白上存在2个共同的差异位点,分别是N115位和N206位。结论:在云南省芒市地区设置的哨兵山羊血液标本分离到AKAV,具有重要的流行病学意义。“,”Objective:To study the prevalence of Akabane virus (AKAV) in Yunnan province.Methods:A group of sentinel animals including 5 goats and 10 cattles which were sero-negative for bluetongue virus and AKAV were located in Mangshi, Yunnan province, to monitor arbovirus activity from April to October 2015. The heparin-anticoagulated blood of the animals was collected weekly for arbovirus isolation. Erythrocytes were lysed in distilied water and inoculated onto BHK-21 monolayer for virus isolation. After the cells showed cytopathic effect (CPE), AKAV in the cell cuture were identified by RT-PCR amplification with AKAV S gene segment specific primers and gene sequencing.Results:The result indicated that BHK-21 cell inoculated with the blood from one sentinel goat showed CPE 48 h post inoculation. One day old suckling mice intracerebrally inoculated with the supernatant of the cell culture, they started to become sick and died after 3 days. RT-PCR identification and S gene sequencing showed that the isolate was AKAV (numbered as 16415). The full length of S segment gene of 16415 is 856 nt, encoding 233 amino acids of protein N, and 91 amino acids of protein Ns. Phylogenetic analysis showed that, among the AKAV strains isolated from China and abroad, the newly isolated 16415 was in the same branch with the other viruses in different geographical regions, and their S segment nucleotide sequence have a high homology of 83.8%-97.7%. The further study show that 16415 and the AKAV isolated from the domestic bamboo rat in Guangxi in 2013 have a closest realtionship in evolution, the nucleotide sequence homology was 97.7%, and amino acid homology is 99.6%. Compared with the Japanese OBE-1 strain, the amino acids of protein N of 16415 and the AKAVs, isolated from banboo rat in Guangxi and n Anopheles vagas in Mangshi of Yunnan province of China, have two common diverse amino acids loci located at the 115th and 206th sites respectively.n Conclusions:It is concluded that AKAV was newly isolated from goat in Mangshi of Yunnan province, which may have important epidemiological significance.
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