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Giardia lamblia, a prevalent human pathogen and one of the lineages that branched earliest from prokaryotes, can be infected with a double-stranded RNA virus, giardiavirus (GLV). The 6,277-bp viral genome has been previously cloned (A.L. Wang, H.-M. Yang, K.A. Shen, and C.C. Wang, Proc. Natl. Acad. Sci. USA 90:8595-8599, 1993; C.-H. Wu, C.C. Wang, H.M. Yang, and A.L. Wang, Gene, in press) and was converted to a transfection vector for G. lamblia in the present study. By flanking the firefly luciferase gene with the 5' and 3' untranslated regions (UTRs) of the GLV genome, transcript of the construct was synthesized in vitro with T7 polymerase and used to transfect G. lamblia WB trophozoites already infected with GLV (WBI). Optimal electroporation conditions used for the transfection were set at 1,000 V/cm and 500 microF, which resulted in expression of significant luciferase activity up to 120 h after electroporation. Furthermore, the mRNA and the antisense RNA of the luciferase gene were both detected by reverse transcription and PCR from 6 to 120 h postelectroporation, whereas no antisense RNA of luciferase was observed in the electroporated virus-free Giardia WB trophozoites. The mRNA of luciferase was detectable in the virus-free trophozoites by reverse transcription and PCR only up to 20 h after the electroporation, indicating that the introduced mRNA was replicated only by the viral RNA-dependent RNA polymerase inside the WBI cells. This expression of luciferase was dependent on the presence of UTRs on both ends of the viral genome transcript, including a putative packaging site that was apparently indispensable for luciferase expression. This is the first time that a viral vector in the form of mRNA URTs has been successfully used in transfecting a protozoan.

The linear double-stranded RNA (dsRNA) genome of giardiavirus (GLV) was estimated to be 6100 nucleotides (nt) [Wang et al., Proc. Natl. Acad. Sci. USA 90 (1993) 8585-8599]. As the 5'- and 3'-untranslated regions (UTR) of viral genomes are known to contain critical information for viral replication, we reexamine the sequences at all four termini of GLV dsRNA by (i) direct RNA sequencing with RT, (ii) tailing GLV dsRNA with UTP or CTP in addition to ATP in 3' rapid amplification of cDNA ends (RACE) and (iii) adding poly(dG) to the products of primer extension in 5'-RACE. The results confirmed the reported sequence for the 5'-terminus of the GLV sense strand RNA, but uncovered an additional 177 nt at the 3'-terminus. The new study also showed conclusively that there are no protruding overhangs at either terminus of GLV dsRNA.

The double-stranded RNA genome of giardiavirus (GLV) has only two large open reading frame (ORFs). The 100-kDa capsid polypeptide (p100) is encoded by ORF1, whereas the only other viral polypeptide, the 190-kDa GLV RNA-dependent RNA polymerase (p190), is synthesized as an ORF1-ORF2 fusion protein by a (-1) ribosomal frameshifting. Edman degradation revealed that p100 was N-terminally blocked except for 2 to 5% of it that showed free N terminus starting from amino acid residue 33 of ORF1. Studies using antiserum targeted against amino acid residues 6 to 27 indicated that this region (NT) is absent from viral p100 and p190, while pulse-labelling experiments showed that NT is present in nascent p100 synthesized in GLV-infected Giardia lamblia but removed subsequently. In contrast, this region was retained in the two viral proteins synthesized in vitro, and it was not removed upon prolonged incubation or inclusion of microsomal fraction in the in vitro translation reaction mixtures. These results suggest that endoplasmic reticulum is not involved in the protein processing and that the precursors of p100 and p190 are incapable of cleaving themselves or each other. This specific cleavage was reproduced when lysates from GLV-infected G. lamblia were added, but not those from uninfected cells. The cleavage activity was relatively insensitive to phenylmethylsulfonyl fluoride, but it was inhibitable by leupeptin or E-64, two known specific inhibitors of cysteine protease. The possible origin of this processing activity is discussed.

Giardia lamblia virus (GLV) is a small nonenveloped double-stranded RNA virus that infects specifically the parasitic protozoan G. lamblia. Among the many collected strains of G. lamblia, a few turn out to be highly resistant to the virus infection. Two of these strains, Ac and JH, were subjected to electroporation with the RNA from GLV-infected G. lamblia WB strain. Subsequent studies indicated the presence of GLV double-stranded RNA and GLV protein in the electroporated and propagated cells. Virus particles, released by the transfected cells into the culture medium, were capable of infecting the virus-sensitive G. lamblia WB strain. When the WB cells were incubated with GLV at 4 degrees C and treated with the bifunctional cross-linking reagent disuccinimidyl suberate, little GLV protein was detectable inside the cells by immunofluorescent staining. However, patches of fluorescent granules were found on the membrane surface of the cells, suggesting cross-linking of the viruses with a certain membrane component(s). Similar treatment of the resistant strains Ac and JH showed no fluorescence either inside or outside of the cells. Two other closely related parasitic protozoa, Tritrichomonas foetus and Trichomonas vaginalis, cannot be infected by GLV via either viral infection or RNA transfection. The [35S]cysteine-labeled protein profiles in Triton X-114 extracts of G. lamblia WB, Ac, and JH were compared. The profile of the WB strain differs clearly from that of Ac and JH. It remains to be seen, however, whether this difference is related at all to the different susceptibilities to GLV infection.

Giardiavirus is a small, nonenveloped virus comprising a monopartite double-stranded RNA genome, a major protein of 100 kDa, and a less abundant polypeptide of 190 kDa. It can be isolated from the culture supernatant of Giardia lamblia, a parasitic flagellate in human and other mammals, and efficiently infects other virus-free G. lamblia. A single-stranded copy of the viral RNA can be electroporated into uninfected G. lamblia cells to complete the viral replication cycle. Giardiavirus genomic cDNA of 6100 nt was constructed and its sequence revealed the presence of two large open reading frames that are separated by a -1 frameshift and share an overlap of 220 nt. The 3' open reading frame contains all consensus RNA-dependent RNA polymerase sequence motifs. A heptamer-pseudoknot structure similar to those found at ribosomal slippage sites in retroviruses and yeast killer virus was identified within this overlap. Immunostudies using antisera against synthesized peptides from four regions in the two open reading frames indicated that the 100- and 190-kDa viral proteins share a common domain in the amino-terminal region. But the 190-kDa protein makes a -1 switch of its reading frame beyond the presumed slippage heptamer and is therefore a -1 frameshift fusion protein similar to the gag-pol fusion protein found in retroviruses.

The subcellular distribution of Giardia lamblia virus RNA in infected G. lamblia trophozoites was examined by in situ hybridization using biotinylated DNA probe and riboprobe. In G. lamblia Portland I strain, which is chronically infected by G. lamblia viruses, the viral RNA was detected in the cytoplasm as well as in the twin nuclei. When riboprobe was used to examine the course of virus infection in WB strain, accumulation of viral RNA was detected only in the cytoplasm prior to the first 72 hr of infection. Using DNA probe, further accumulation of viral RNA in increasing number of cells occurred after the 72nd hr of infection, with the RNA found in both the cytoplasm and nuclei. Eventually, the cell nuclei showed damaged morphology that deteriorated rapidly toward the final stage of infection. These observations indicate that early phase of viral RNA replication may take place in the cytoplasm of infected G. lamblia, but the nuclei are also involved during the late phase of viral replication.

The presence or absence of the Giardia lamblia double-stranded RNA virus (GLV) was surveyed among 38 axenic isolates of G. lamblia derived from both humans and animals. Of the 28 isolates lacking the virus, 19 could readily be infected by the virus. The remaining 9 isolates proved to be resistant to GLV infection even when the ratio between virus to parasite reached as high as 10(6) to 1. Evidence is also presented indicating that there are at least two "Portland 1" strains being used by the current scientific community, one containing the virus and the other lacking the virus.

The double-stranded RNA virus-like particles, found among several independent isolates and cloned strains of Giardia lamblia, have previously been reported to be spheres of 35 nm with a genome of 7 kilobase pairs and a major protein of 100 kDa. The virus is capable of infecting certain virus-free isolates of G. lamblia. Antisera raised in mice against the intact virus did not react with the double-stranded RNA, but reacted strongly with the 100 kDa protein in Western blots. Preincubation of the virus with antisera abolished viral infectivity, whereas the antisera against double-stranded RNA showed only a weak blocking effect. Inclusion of the antiviral sera in the cultures of virus-infected G. lamblia at 10(3)-fold dilution resulted in elimination of the virus from the protozoa. Apparently, the 100 kDa protein is necessary for the initiation of viral infection and possibly subsequent assembly or replication of viral progeny particles.

The dsRNA virus which infects some strains of Giardia lamblia has been purified and characterized with respect to its effect on growth of the parasite. Extensive purification of the virus from G. lamblia growth medium was accomplished by Millipore filtration and two successive CsCl gradient centrifugations. The purified virus possessed a single major protein species of 100,000 molecular weight. Effects of the extensively purified virus on growth of the virus-free parasite were studied. A cloned WB strain, sensitive to the viral infection, and a cloned E-9/M strain, resistant to the infection, were studied. With the WB strain, infection can occur at a ratio as low as 10 viral particles per organism. As the virus to parasite ratio increased, the rate of growth of the parasite decreased and the percentage of parasites not adhering to the culture tube wall also increased. These nonadhering cells, which differed from the nonadhering cells under normal growth conditions, were unable to divide. They contained an average number of 500,000 viral particles per cell which may be the threshold intracellular density of viral particles arresting the growth of G. lamblia. The results also suggest that the specific consequence of viral infection, even at extremely high multiplicity of infection, is not lysis of G. lamblia trophozoites but cessation of growth.

An isolate of Giardia lamblia infected with the double-stranded RNA virus (GLV) has two major species of RNA that are not present in an uninfected isolate. One of these species is the previously characterized double-stranded RNA genome of GLV (1). The second species of RNA appears to be a full length copy of one strand of the double-stranded RNA genome. This full length single-stranded RNA is not present in viral particles isolated from the growth medium. The cellular concentration of the single-stranded RNA changes during exponential and stationary phases of cell growth in a fashion consistent with a viral replicative intermediate or mRNA. The single-stranded species does not appear to be polyadenylated.

Nucleic acid samples purified from trophozoites of Giardia lamblia Portland I strain contain an ethidium-stainable band that comigrates with 7.0 kilobase DNA in agarose gel electrophoresis. The band was degradable by alkali, ribonuclease A and ribonuclease T1, but the susceptibility toward the ribonucleases decreased with increasing ionic strength, suggestive of double-stranded RNA (dsRNA). This identification was confirmed by electron micrographs of the purified samples, which showed linear double-stranded structures with an estimated average length of 1.5 micron. In crude homogenates of G. lamblia, this dsRNA was protected against added ribonuclease A but disappeared upon adding sodium dodecyl sulfate or proteinase K. Differential centrifugations suggested an association of the dsRNA with the nuclear fraction, but it was freed to the 109,000 X g pelletable fraction with increasing homogenization. The dsRNA was purified by CsCl buoyant density gradient centrifugations in a distinct band with a rho value of 1.368 g ml-1. Electron microscopy revealed spherical virus-like particles (VLP) with a diameter of 33 nm. VLP of similar shape and size were also identified in the nuclei of sectioned G. lamblia trophozoites. VLP yield a major protein with an estimated molecular weight of 66,000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis. VLP are abundant in the culture media of stationary-phase G. lamblia Portland I strain and are able to infect the G. lamblia WB strain, which is free of the virus. There is no sequence homology between the dsRNA and the nuclear DNA of G. lamblia and thus no apparent integration of viral genome into host DNA.