Giardiavirus: an update.

Giardiavirus is the only virus that infects Giardia duodenalis, a highly prevalent parasite worldwide, especially in low-income and developing countries. This virus belongs to the Totiviridae family, being a relative of other viruses that infect fungi and protozoa. It has a simple structure with only two proteins encoded in its genome and it appears that it can leave the cell without lysis. All these characteristics make it an interesting study model; however, its research has unfortunately made little progress in recent years. Thus, in this review, we summarize the currently available data on Giardiavirus, from their structure, genome and main proteins, to the uses that have been given to them and the possible health applications for the future.

Giardia duodenalis: genetic recombination and its implications for taxonomy and molecular epidemiology.

Traditionally, species within the Giardia genus have been considered as eukaryotic organisms that show an absence of sexual reproduction in their simple life cycles. This apparent lack of sex has been challenged by a number of studies that have demonstrated (i) the presence in the Giardia duodenalis genome of true homologs of genes specifically involved in meiosis in other eukaryotes, and their stage-specific expression; (ii) the exchange of genetic material in different chromosomal regions among human isolates of the parasite; (iii) the fusion between cyst nuclei (karyogamy) and the transfer of genetic material (episomal plasmids) between them. These results are pivotal for the existence of sexual recombination. However, many details of the process remain elusive, and experimental data are still scarce. This review summarizes the experimental approaches and the results obtained, and discusses the implications of recombination from the standpoint of the taxonomy and molecular epidemiology of this widespread pathogen.

Giardia canis: ultrastructural analysis of G. canis trophozoites transfected with full length G. canis virus cDNA transcripts.

Giardia canis virus (GCV) is a double-stranded RNA (dsRNA) virus of the family Totiviridae. In this study, the full length cDNA of the G. canis virus was constructed in pPoly2/sfinot vector and RNA was transcribed in vitro. Virus-free G. canis trophozoites were transfected with in vitro transcribed GCV RNA by electroporation. Transfected trophozoites were cultured for 12, 24, 36, 48, 60, or 72h post transfection for analysis. The ultrastructures of the transfected trophozoites were determined by transmission electron microscopy. The viral particles were detectable sporadically in the cytoplasm as early as 24h post transfection, but became evident and wide-spread 36h post transfection. The number of viral particles increased dramatically from 48 to 60h. Viral particles were released into the culture medium starting at about 60h and detectable in nuclei 72h post transfection. Severe vacuolization was seen in transfected G. canis trophozoites as early as 36h post transfection and persisted throughout the course of this study. The results of the present study indicate that in vitro transcribed GCV transcripts were capable of infecting Giardia trophozoites, apparently replicated and packaged into mature infectious viral particles which were released from the host.

Giardia lamblia: stable expression of green fluorescent protein mediated by giardiavirus.

Giardia lamblia, an early diverging eukaryote that infects several species including humans and a major agent of water-borne diarrhea throughout the world, can be infected with a double-stranded RNA virus, giardiavirus (GLV). A chimeric GLV cDNA and green fluorescent protein (GFP) according to the cis-acting signals of the GLV genome required for expression of foreign gene was constructed and its in vitro transcript was electroporated into GLV-infected G. lamblia trophozoites, GFP was expressed transiently. pGDH5/NEO/GLV was constructed by combining the neomycin resistance cassette in which the neomycin phosphotransferase gene was flanked by Giardia glutamate dehydrogenase (GDH) uncoding regions and the transcription cassette in which the chimera of GLV cDNA and GFP was located downstream from GDH gene promoter on a single plasmid. This plasmid was electroporated into G. lamblia and the transfectants persistently expressed GFP under G418 selection. This stable transfection system should provide a valuable tool for genetic study of G. lamblia.

Amplification, expression, and packaging of a foreign gene by giardiavirus in Giardia lamblia.

Giardia lamblia is an intestinal protozoan parasite and one of the earliest eukaryotic divergents. The trophozoite multiplies via asexual binary fission and lacks all natural means of lateral gene transfer. A system is developed here for long-term expression of a foreign gene in this organism by exploiting recombinant virions derived from the giardiavirus (GLV), a double-stranded RNA virus that infects many Giardia isolates. An in vitro transcript of the cloned GLV cDNA, comprising the firefly luciferase-encoding region flanked by 5' and 3' fragments of GLV positive-strand RNA, was electroporated into GLV-infected trophozoites. Luciferase activity in electroporated cells peaked on day 2 at levels 6 orders of magnitude above background. Expression of this foreign gene remained at 80% of its peak level after 30 days in the absence of selective pressure. The chimeric RNA was replicated as double-stranded RNA and packaged into virus-like particles. The recombinant virions were partially purified from the wild-type helper virus by CsCl equilibrium density-gradient centrifugation and used to superinfect Giardia trophozoites. At multiplicities of infection of 100 or higher, these chimeric virions were able to initiate new rounds of expression of luciferase activity in the superinfected cells. Thus, the engineered virion can be successfully used to introduce and efficiently express a heterologous gene in this eukaryotic microorganism.