DOUBLE-STRANDED RNA VIRUSES IN TRICHOMONAS & GIARDIA

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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.