革螨和恙螨体内汉坦病毒增殖与定位的分子生物学研究

目的 研究汉坦病毒在革螨、恙螨体内增殖、定位及与传播肾综合征出血热（HFRS）的关系。方法 采用聚合酶链反应（PCR）检测螨体内HV－RNA和基因分型;Vero-E6细胞培养检测TCID50／ml滴度;原位RT－PCR分子杂交法检测HV－RNA在螨体内分布和定位。结果 用PCR技术从革螨、恙螨体内检测到HV－RNA;取革螨、恙螨幼虫、若虫定期做HV TCID50／ml滴度,证明HV在革螨、恙螨体内可经期传播,并有增殖现象;用原位RT－PCR分子杂交法在革螨、恙螨的中肠上皮细胞和卵巢细胞内检测到HV－RNA阳性颗粒;用PCR分型证明从同一疫区鼠、螨、病人所分离的HV的基因型一致。结论 研究结果为革螨、恙螨作为HFRS的媒介提供了分子水平的证据,对HFRS的流行病学和预防具有重要的理论意义和实用价值。

Proliferation and location of Hantaan virus in gamasid mites and chigger mites,a molecular biological study

OBJECTIVE: To study the proliferation and location of Hantaan virus (HV) in gamasid mites and chigger mites and the significance of gamasid mites and chigger mites as vectors of transmission of hemorrhagic fever with renal syndrome (HFRS). METHODS: Gamasid mites collected from the nests of wild rodents and chigger mites collected from the bodies of wild rodents and thick growth of grass in the field were raised. Two oligonucleotide primers were developed based on the gene fragments of cDNA of HV 76 - 118 strain to be used in RT-PCR. RNA was extracted from the suspension of the gamasid mites from the nests where rodents no HV had been found in whose lungs lived and from the unfed larvae of chigger mites, being formed groups of 5, 10, 30, or 50 individuals. RT-PCR was conducted to detect the HV-RNA in such suspension. The larvae, nymphs, and imagines of both kinds of mites were ground to make suspension at an interval of 20 days. Vero-E(6) cells were inoculated to measure the titer of 50% tissue culture infective dose (TCID(50))/ml of HV. Frozen sections of larvae, nymphs, and imagines of both kinds of mites were made. RT-PCR and in situ hybridization were conducted to detect the distribution of HV-positive particles. Monoclonal antigen technique was used to compare the antigenicity of the HV-RNA from the rodents, mites, and patients from the same epidemic areas. RESULTS: HV-RNA was detected in gamasid mites and chigger mites. Except in the larvae of chigger mites 60 days after collection, titers of HV were detected and increased gradually in mites at different stages of life cycle. HV-RNA positive particles were detected in the epithelial cells of midgut and ovary, with the signal denser and more numerous in nymphs than in larvae. The genotypes of HV from rodents, mites and patients in the same endemic areas were identical: HTN type virus. CONCLUSION: HV can be transmitted transstadially and proliferated in mites, gamasid mites and chigger mites play the role of vectors of transmission for HFRS.