DOUBLE-STRANDED RNA VIRUSES IN TRICHOMONAS AND GIARDIA

毛滴虫病和贾第虫病是两种流行的人体寄生虫病 世界上病原体阴道毛滴虫和贾第虫 这两种疾病的病原都是厌氧鞭毛虫 无性繁殖，不为研究人员提供遗传信息。 最近，我们的实验室首次发现了两种独特的 双链（ds）RNA病毒在两种生物体中。霸王阴道毛 病毒（TVV）是非感染性的，在细胞内繁殖，并与 表达至少一种T.流浪汉 共和党兰氏病毒（GLV）可感染无病毒的G.兰氏滋养体 通过与细胞表面的假定受体结合，但感染 对细菌的生长或毒力均无不良影响。 寄生虫在GLV中发现RNA依赖的RNA聚合酶活性。（+） 鉴定了7 kb病毒dsRNA基因组的单链RNA（SS）， 从感染的细胞中纯化，这有助于克隆和 从SS的5 ′-端开始测定1.6kb的cDNA序列。纯化的SS为 成功地引入G.通过电穿孔将Lamblia滋养体， 然后将其转化为GLV，增殖并从细胞中挤出， dsRNA在病毒颗粒中形成，从而实现G. 兰布利亚目前，我们正计划在多个 电穿孔寄生原生动物，并监测细胞内GLV 形成转染的迹象。克隆的5 ′-SS片段的cDNA， 其中最有可能包含GLV基因组的调控单位，将是 用外源基因标记，例如，氯霉素乙酰转移酶或 腺苷脱氨酶（潜在的抗贾第虫作用，见正文）。 电穿孔法将体外转录物导入GLV感染的 G.预计兰布利亚将从病毒中获得所需的帮助。的 转录物可以转化为dsRNA，包封在病毒颗粒中， 在G. Lamblia，从而达到表达外国的目的 基因转染。在这一点上，这种成功的前景是 相当乐观。将克隆GLV基因组全长cDNA 也追求。这一努力的成功将使外源基因的表达在 G.没有辅助病毒的兰布拉病毒GLV发展的时间进程， 将通过核酸原位杂交监测GLV的移动 和免疫金染色。转录酶和复制酶的功能， 将分析病毒RNA聚合酶，以了解 GLV复制。GLV的推定受体将被纯化， 克隆、测序并在酵母中表达以用于GLV感染。最后我们 我会在荃湾重复大部分计划中的研究，以便我们可以 比较这些知识，以便更好地了解T. vagelis和G.兰布利亚

Trichomoniasis and giardiasis are two prevalent human parasitic diseases in the world. The causative agents Trichomonas vaginalis and Giardia lamblia for the two diseases are both anaerobic flagellated protozoa multiplying asexually and providing no genetic information for researchers. Recently, our laboratory identified, for the first time, two unique double-stranded (ds) RNA viruses in the two organisms. The T. vaginalis virus (TVV) is noninfectious, propagated intracellularly, and associated with the expression of at least one major surface antigen of T. vaginalis. The G. lamblia virus (GLV) can infect virus-free G. lamblia trophozoites by binding to the putative receptors on the cell surface, but the infection exerts no adverse effect on either the growth or the virulence of the parasite. RNA-dependent RNA polymerase activity was found in GLV. The (+) single-stranded RNA (SS) of the 7 kb viral dsRNA genome was identified and purified from the infected cells, which facilitated the cloning and sequencing of a 1.6 kb cDNA of SS from its 5'-end. The purified SS was successfully introduced into G. lamblia trophozoites by electroporation, which was then converted to GLV, multiplied and extruded from the cells in dsRNA form in virus particles; thus achieving stable transfection of G. lamblia. At present, we are planning on introducing SS into a variety of parasitic protozoa electroporatically, and monitoring intracellular GLV formation for signs of transfection. The cloned cDNA of the 5'-SS fragment, which most likely contains the regulatory unit of GLV genome, will be tagged with a foreign gene, e.g., chloramphenicol acetyltransferase or adenosine deaminase (for potential antigiardiasis action, see text). Electroporatic introduction of the in vitro transcripts into GLV-infected G. lamblia is expected to receive the needed help from the virus. The transcripts may be converted to dsRNA, encapsulated in virus particles and expressed in G. lamblia, thus achieving the purpose of expressing foreign genes by transfection. The prospect for such a success at this point is quite optimistic. Cloning of the full-length cDNA of GLV genome will be also pursued. Success in this effort will allow foreign gene expression in G. lamblia without the helper virus. Time courses of GLV development and movements of GLV will be monitored by nucleic acid in situ hybridizations and immunogold stainings. The transcriptase and replicase functions of the viral RNA polymerase will be analyzed for understanding the mechanism of GLV replication. The putative receptor of GLV will be purified and the gene cloned, sequenced and expressed in yeast for GLV infection. Finally we shall repeat on TW most of the planned studies on GLV so that we can compare these knowledges for better understanding of gene regulation in T. vaginalis and G. lamblia.