一株牛源阿卡斑病毒的分离鉴定

Isolation and Identification of Akabane Virus from Cattle in China

阿卡斑病毒(Akabane virus,AKV)是能引起牛、绵羊、山羊流产、早产、新生胎儿畸形的虫媒性RNA病毒。为了解家畜虫媒病毒在我国西南边境地区的分布和流行情况,本研究对中缅边境西盟县的52份牛抗凝血和140份血清(牛70份、羊70份)中的蓝舌病病毒(Bluetongue virus,BTV)、鹿流行性出血热病毒(Epizootic hemorrhagic disease virus,EHDV)、AKV等虫媒病毒进行检测与分离,通过ELISA及qRT-PCR方法检测病毒,通过核酸阳性抗凝血接种BHK细胞传代以分离病毒,通过设计特异性引物,扩增分离毒株S基因721bp片段及M基因816bp片段,通过克隆测序及中和试验以鉴定病毒,最终从38号牛的抗凝血中分离到一株AKV,TCID50为10-3.5/0.1mL,经比对,分离株S片段与日本KS-2/Mo/06毒株亲缘关系最近,核苷酸同源性为97.66%,M片段与中国DHL10M110毒株亲缘关系最近,核苷酸同源性为96.56%。本研究首次报告了从云南边境地区牛群中分离到AKV,证实了西南边境存在AKV的流行,为AKV在我国的流行病学和边境地区疫病风险防控提供了重要参考及有力依据。

Akabane virus(AKV)is an arbovirus that can cause abortion,premature birth and congenital deformities such as arthrogryposis-hydranencephaly syndrome in cattle,sheep and goats.In order to investigate the distribution and prevalence of arbovirus in southwest China,bluetongue virus(BTV),epizootic hemorrhagic disease virus(EHDV)and AKV were detected and isolated from 52 bovine anticoagulants and 140 serum(70 for cattle and 70 for sheep)in Ximeng County,Yunnan Province,China.Nucleic acid-positive blood samples were inoculated with BHK cells to isolate viruses,isolated strains were identified by enzyme-linked immunosorbent assays and amplification by reverse transcription-polymerase chain reaction.Segment S(Seg-S)and Segment M(Seg-M)were amplified with specific primers,then cloned and sequenced.The serum neutralization test and molecular analysis showed that V38/YN/2018 strain was AKV.TCID50 was 10-3.5 per 0.1 mL.Sequencing analyses demonstrated that the Seg-S of the V38/YN/2018 strain showed 97.66%nucleotide identity with the Japanese strain(KS-2/Mo/06),whereas Seg-M showed 96.56%nucleotide identity with the Chinese strain(DHL10 M110).This is the first report of isolation of AKV from cattle in the border areas of Yunnan Province,China.Our data provide a basis for epidemiologic investigation,as well as risk prevention and control of the AKV in China.