大兴安岭全沟硬蜱携带哺乳动物腮腺炎病毒5型的分离鉴定

Isolation and identification of mammalian rubulavirus type 5carried by Ixodes persulcatus from the Greater Khingan Mountains

为了明确黑龙江省大兴安岭地区媒介生物蜱携带病原的本底情况,本研究采集黑龙江省大兴安岭呼玛县、漠河县、塔河县、白桦乡、三卡乡、塔列图河、五叉沟和加北乡各地区的全沟硬蜱作为研究对象,利用病毒宏基因组学分析方法,经Illumina HiSeq2000高通量测序,共得到78091166条读长,经生物信息学分析,结果显示,其中涵盖的序列主要包括布尼亚病毒、巴库病毒、哺乳动物腮腺炎病毒和汉坦病毒等,并进一步对哺乳动物腮腺炎病毒5型进行了分离鉴定,将通过PCR检测为哺乳动物腮腺炎病毒5型阳性的样品接种PK15细胞分离培养,结果显示出现明显的细胞病变,培养第5 d病毒滴度达1×10^(5.7)TCID^(50)/mL。盲传4代后经PCR鉴定为哺乳动物腮腺炎病毒5型阳性。进一步进行电镜观察,可见病毒粒子直径为50 nm~60 nm,完整的病毒粒子呈球形,有囊膜,且间接免疫荧光试验检测为阳性。经PCR扩增哺乳动物腮腺炎病毒5型全基因组序列并测序分析,结果显示该病毒的全长基因组序列与哺乳动物腮腺炎病毒5型的序列相似度高达96%,进一步验证了病毒宏基因组学分析结果。本研究首次从黑龙江省大兴安岭地区全沟硬蜱中分离得到一株哺乳动物腮腺炎病毒5型,为该地区蜱携带哺乳动物腮腺炎病毒5型的流行病学调查奠定了基础。

To investigate prevalence of virus transmitted by ticks at the Greater Khingan Mountain area of Heilongjiang province,virus burden were detected in Ixodes persulcatus collected from Huma,Mohe,Tahe,Baihua,Sanka,Taletu River Wuchaogou and Jiabie in this study.Metagenomic analysis was used to explore viruses and 78091166 reads were obtained through high-throughput sequencing technology.Bioinformatic analysis indicated that specific sequences of many viruses,including Bunya virus,Baku virus,mammalian rubulavirus,Hanta virus etc.A mammalian rubulavirus 5 was further isolated and identified by inoculating the PCR-positive samples into PK15 cells.Obvious cytopathic effect(CPE)could be observed in the cells infected with the virus.The titer of the virus reached to 1×10^(5.7)TCID_(50)/mL on the 5th day.Spherical virus particles with envelope and diameter ranging from 50nm to 60nm were observed under transmission electron microscopy(TEM).The fact that the genome sequence of the isolated mammalian rubulavirus 5 share 96%similarity with a reference strain,further proved the data of our metagenomic analysis.