云南省澜沧江下游地区虫媒病毒的调查研究

目的 了解云南澜沧江下游地区自然界虫媒病毒流行情况。方法 从云南澜沧江下游地区(澜沧县和思茅市)所属乡村采集蚊虫,分类后保存于液氮。经消毒、研磨、离心等处理后接种组织培养细胞分离病毒,并进行初步鉴定。结果从233份标本中,分离到22株致C6/36细胞病变的病毒,表现为细胞圆化、聚集、脱落。分离阳性率为9.4％(22/233)。经鉴定10株对乙醚和5’-磺脱氧尿苷抵抗,为无包膜双链RNA病毒,核酸电泳为12节段RNA病毒。9株对乙醚敏感,对5’-碘脱氧尿苷抵抗,为有膜RNA病毒,核酸电泳为10条带。Colti病毒95-75单克隆抗体与新分离的12节段RNA病毒的酶联免疫吸附试验(ELISA)和免疫荧光试验呈阳性反应,而与10节段病毒呈阴性反应。3株病毒为对乙醚敏感,其中1株被乙型脑炎病毒A2株免疫腹水中和,并与A2免疫腹水荧光试验呈阳性反应;1株(云南92-4)与布尼亚病毒组特异免疫腹水的荧光试验和ELISA试验均为强阳性,而与甲病毒组特异性免疫腹水及黄病毒组的乙型脑炎病毒免疫腹水均呈阴性反应;经负染在电镜下观察毒粒呈圆形,直径约为(87.00±0.05)nm。外层可见表面突起。结论 从云南澜沧江下游地区采集的蚊标本分离到10株Colti病毒,9株环状病毒,1株为乙脑病毒,1株为布尼亚病毒,1株尚在研究中。

Investigation of Arboviruses in Lancang river down-stream area in Yunnan province

Objective To investigate the epidemic state of arboviruses in the down-stream area of Lancang river in Yunnan province. Methods Mosquitoes were collected from Lancang river dowm-stream area (including Lancang county and Simao city) during summer in 1998 and stored in liquid nitrogen after classification. The mosquito pools were homogenized, and centrifuged, then the supernatant was inoculated into C6/36 cells for virus isolation. New isolates were identified by neutralization test (NT), ELISA, immunoflurescence assay(IFA) and polyacrylamid gel electrophoresis ( PAGE) . Result Totally 22 isolates of arbovirus were obtained from 233 mosquito pools by inoculation of C6/36 cells and positive rate of the isolation was 9.4% . Ten strains were resistant to both ether and 5'-IDU. So they were non-enveloped double-stranded (ds) RNA virus. Twelve segmented RNAs were shown by PACE and PACE profiles from the ten strains were 6-6 with minor variation. The isolates can be neutralized by immunized mouse ascites fluid of BJ95-75 strains of coltivirus by NT, and reacted with monoclonal antibody against BJ95-75 by ELISA. These viruses were identified as coltivirus. Nine isolates were sensitive to ether and resistant to 5'-IDU. So they were non-enveloped RNA viruses. PAGE showed 10 segmented RNA, and they were identified to be orbiviruses. Three isolations were sensitive to ether. One of them can be neutralized with JEV A2 strain antibody by NT and was positive to the homologous antibody by IFA. It was identified being strain of JE virus. One strain(YN92-4) can be reacted with anti-bunyavirus group specific immune ascites fluid by both IFA and EIISA, but reacted neither with anti-alpha virus group, nor with anti-flavivirus group JE virus ascites fluid. The virions are spherical and about 87nm in diameter with surface projections by negative staining. Conclusion Twenty-two isolates have been obtained from wild caught-mosquitoes of Lancang river down- stream area in Yunnan province. Among them ten, nine ,one and one were identified as coltivirus, orbivirus, JE virus and bunyavirus, respectively. One is under identification. This is the first report on bunyavirus isolated from mosquitoes in China.