Three-dimensional structure of a protozoal double-stranded RNA virus that infects the enteric pathogen Giardia lamblia.

UNLABELLED: Giardia lamblia virus (GLV) is a small, nonenveloped, nonsegmented double-stranded RNA (dsRNA) virus infecting Giardia lamblia, the most common protozoan pathogen of the human intestine and a major agent of waterborne diarrheal disease worldwide. GLV (genus Giardiavirus) is a member of family Totiviridae, along with several other groups of protozoal or fungal viruses, including Leishmania RNA viruses and Trichomonas vaginalis viruses. Interestingly, GLV is more closely related than other Totiviridae members to a group of recently discovered metazoan viruses that includes penaeid shrimp infectious myonecrosis virus (IMNV). Moreover, GLV is the only known protozoal dsRNA virus that can transmit efficiently by extracellular means, also like IMNV. In this study, we used transmission electron cryomicroscopy and icosahedral image reconstruction to examine the GLV virion at an estimated resolution of 6.0 Å. Its outermost diameter is 485 Å, making it the largest totivirus capsid analyzed to date. Structural comparisons of GLV and other totiviruses highlighted a related "T=2" capsid organization and a conserved helix-rich fold in the capsid subunits. In agreement with its unique capacity as a protozoal dsRNA virus to survive and transmit through extracellular environments, GLV was found to be more thermoresistant than Trichomonas vaginalis virus 1, but no specific protein machinery to mediate cell entry, such as the fiber complexes in IMNV, could be localized. These and other structural and biochemical findings provide a basis for future work to dissect the cell entry mechanism of GLV into a "primitive" (early-branching) eukaryotic host and an important enteric pathogen of humans. IMPORTANCE: Numerous pathogenic bacteria, including Corynebacterium diphtheriae, Salmonella enterica, and Vibrio cholerae, are infected with lysogenic bacteriophages that contribute significantly to bacterial virulence. In line with this phenomenon, several pathogenic protozoa, including Giardia lamblia, Leishmania species, and Trichomonas vaginalis are persistently infected with dsRNA viruses, and growing evidence indicates that at least some of these protozoal viruses can likewise enhance the pathogenicity of their hosts. Understanding of these protozoal viruses, however, lags far behind that of many bacteriophages. Here, we investigated the dsRNA virus that infects the widespread enteric parasite Giardia lamblia. Using electron cryomicroscopy and icosahedral image reconstruction, we determined the virion structure of Giardia lamblia virus, obtaining new information relating to its assembly, stability, functions in cell entry and transcription, and similarities and differences with other dsRNA viruses. The results of our study set the stage for further mechanistic work on the roles of these viruses in protozoal virulence.

A real-time PCR for the detection of infectious myonecrosis virus in penaeid shrimp.

Infectious myonecrosis virus (IMNV) is a recently observed shrimp virus, which threats the cultured Litopenaeus vannamei and can cause huge economic loss in shrimp farming industry. The specific aim of this study was to develop a new sensitive real-time PCR method for the specific detection of shrimp IMNV. A real-time PCR assay with a pair of primers to specifically amplify a 101bp IMNV cDNA fragment and a corresponding TaqMan probe was developed, which shown to be specific for IMNV without cross reaction with DNA samples prepared from four other shrimp viruses including white spot syndrome virus (WSSV), hepatopancreatic parvovirus (HPV), monodon baculovirus (MBV), and infectious hypodermal and haematopoietic virus (IHHNV). The method could detect as low as one single copy of IMNV plasmid cDNA.

Purification of infectious myonecrosis virus (IMNV) in species of marine shrimp Litopenaeus vannamei in the State of Ceará.

In Brazil, shrimp farming has been developed most intensely in the Northeast Region. Recently, however, exporters have become concerned over the appearance of Infectious Myonecrosis (IMN), the etiological agent of which is a virus called Infectious Myonecrosis Virus (IMNV). Although IMNV has been characterized extensively, purification methods are complicated to reproduce and very expensive. The objective of this study was to purify the IMNV virus using an easy reproductive method and to produce anti-IMNV antibodies to be used in diagnostic methods. Shrimp samples showing symptoms of IMN obtained from two aquaculture farms in Ceará were used for this purpose. IMNV-positive shrimps were macerated in phosphate buffer, pH 7.5, enriched with antioxidants, clarified with chloroform and the supernatant was submitted to differential centrifugation, precipitated using PEG and NaCl and finally loaded on a discontinuous gradient of sucrose. Purified IMNV was submitted to RT-PCR and electrophoresis either in agarose gel or SDS-PAGE, which revealed RNA and protein bands, characteristic of IMNV. IMNV induced humoral immune response in Swiss mice when administered subcutaneously. Anti-IMNV antibodies were identified by ELISA (enzyme-linked immunosorbent assay) and Western blotting methods and produced a response against purified IMNV and the crude extract obtained from the infected shrimp. However, antibodies specific to the crude extract obtained from uninfected shrimp were not detected. This is the first report of IMNV having been purified in Brazil and the first time that specific antibodies against IMNV proteins have been produced. These results suggest that easy methods can be developed to produce specific antiserum for viral diagnosis on a large scale.

Detection and comparison of Giardia virus (GLV) from different assemblages of Giardia duodenalis.

Five assemblages of Giardia duodenalis were identified from cysts in cattle, dog, cat, sheep, and reindeer feces using ribosomal DNA (rDNA) sequencing. Assemblage A was present in cattle and reindeer feces, Assemblages C and D were present in dog feces, Assemblage E was present in cattle and sheep feces, and Assemblage F was present in cat feces. Giardia virus, originally referred to as Giardia lamblia virus (GLV), is a double-stranded RNA virus. Primers designed for the GLV capsid protein gene identified GLV sequences in G. lamblia from a reindeer (Assemblage A) and from a dog (Assemblage C). Two distinct GLV sequences were identified in the dog specimen and 1 sequence was identified in the reindeer specimen. None of these GLV sequences was identical with previously published GLV sequences. It appears that GLVs are genetically diverse and that more than 1 virion can be present in a single sample. Because many of the specimens that contained cysts were found to be negative for GLV, it appears that this test for capsid protein is of limited value for the purposes of detecting G. lamblia.

Double-stranded RNA virus in Korean isolate IH-2 of Trichomonas vaginalis.

In this study, we describe Korean isolates of Trichomonas vaginalis infected with double-stranded (ds) RNA virus (TVV). One T. vaginalis isolate infected with TVV IH-2 evidenced weak pathogenicity in the mouse assay coupled with the persistent presence of a dsRNA, thereby indicating a hypovirulence effect of dsRNA in T. vaginalis. Cloning and sequence analysis results revealed that the genomic dsRNA of TVV IH-2 was 4,647 bp in length and evidenced a sequence identity of 80% with the previously-described TVV 1-1 and 1-5, but only a 42% identity with TVV 2-1 and 3 isolates. It harbored 2 overlapping open reading frames of the putative capsid protein and dsRNA-dependent RNA polymerase (RdRp). As previously observed in the TVV isolates 1-1 and 1-5, a conserved ribosomal slippage heptamer (CCUUUUU) and its surrounding sequence context within the consensus 14-nt overlap implied the gene expression of a capsid protein-RdRp fusion protein, occurring as the result of a potential ribosomal frameshift event. The phylogenetic analysis of RdRp showed that the Korean TVV IH-2 isolate formed a compact group with TVV 1-1 and 1-5 isolates, which was divergent from TVV 2-1, 3 and other viral isolates classified as members of the Giardiavirus genus.

[Establishment of in vitro cultivation of Giardia canis trophozoites infected with Giardia canis virus].

OBJECTIVE: To cultivate a Giardia canis isolate with G. canis virus (GCV). METHODS: Five-day-old Meriones unguiculatus was infected with the cysts of G. canis isolated from dogs in Changchun and purified by sucrose density gradient centrifugation-G1 acid funnel filtration method. Trophozoites were isolated aseptically from the duodenum of the infected rodent after 8 days, then transferred to modified TYI-S-33 medium and cultivated at 37 degrees C. The trophozoites were centrifuged with 3,000 x g, 15 min after liquid nitrogen freeze-thawing three times and the supernatant stained negatively by phosphotungstic acid was observed with transmission electron microscope. RESULTS: G. canis trophozoites which adapted gradually to the environment and grew a cellular monolayer after 14 days were examined by freezing and thawing experiment, purity quotient, stability, biology characteristics and microbial contamination detection. The results demonstrated that a stable G. canis trophozoite cell isolate was established. G. canis virus with icosahedron spherical shape and 36 nm in diameter was observed by electron microscope. CONCLUSION: In vitro cultivation of G. canis trophozoites with GCV is established.

An examination of risk factors associated with beef cattle shedding pathogens of potential zoonotic concern.

The prevalence of three waterborne zoonotic pathogens (Campylobacter sp., Giardia sp. and Cryptosporidium parvum) in rectal faecal samples from a random sample of adult beef cattle was determined. Management factors that may be associated with shedding of these organisms were examined. For Campylobacter sp. prevalence was 5.0%, and the number of females on the farm was positively associated with the proportion that tested positive. For Giardia sp. prevalence was 6.5%, and none of the management factors examined was significantly associated with the proportion in a herd testing positive. C. parvum was identified in 1.1% of samples. The length of calving season and whether any procedures were performed on the calves in the first 2 days of life were positively associated with the proportion that tested positive. We conclude that this sample of adult beef cattle represent a relatively limited threat to water supplies and subsequent disease transmission to humans from these pathogens.

Identification of virus-specific vesicles in Giardiavirus-infected Giardia lamblia.

Giardiavirus (GLV), which infects the parasitic protozoan Giardia lamblia, is a nonsegmented double-stranded (ds) ribonucleic acid (RNA) virus. We previously purified two distinct types of related GLV from infected G. lamblia, and showed differential export of one of the viruses from infected cells. In the present study, fractionation of cell lysate was performed, revealing the presence of viruses in the membranous fraction. Distribution of viral antigens in the infected cells was examined by immunocytochemistry. The signal was enriched in certain regions of the cytoplasm, suggesting that a portion of GLV is confined to certain cellular compartments. A significantly reduced signal was also detected in the nuclei. We directly observed the viruses in the infected cells by electron microscopy. Consistent with previous observations, virus-like particles were clearly observed in some membranous vesicles in the cytoplasm at 48 h postinfection, and virus-like particles were again seen in the cytoplasm and then in the nuclei toward the late phase of virus infection. The virus-associated vesicles and some electron-dense nuclear structures were only observed in virus-infected cells, suggesting that virus infection may induce ultrastructural alteration of G. lamblia.