A potential role for Giardia chaperone protein GdDnaJ in regulating Giardia proliferation and Giardiavirus replication.

BACKGROUND: Giardia duodenalis (referred to as Giardia) is a flagellated binucleate protozoan parasite, which causes one of the most common diarrheal diseases, giardiasis, worldwide. Giardia can be infected by Giardiavirus (GLV), a small endosymbiotic dsRNA virus belongs to the Totiviridae family. However, the regulation of GLV and a positive correlation between GLV and Giardia virulence is yet to be elucidated. METHODS: To identify potential regulators of GLV, we performed a yeast two-hybrid (Y2H) screen to search for interacting proteins of RdRp. GST pull-down, co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) assay were used to verify the direct physical interaction between GLV RdRp and its new binding partner. In addition, their in vivo interaction and colocalization in Giardia trophozoites were examined by using Duolink proximal ligation assay (Duolink PLA). RESULTS: From Y2H screen, the Giardia chaperone protein, Giardia DnaJ (GdDnaJ), was identified as a new binding partner for GLV RdRp. The direct interaction between GdDnaJ and GLV RdRp was verified via GST pull-down, co-immunoprecipitation and BiFC. In addition, colocalization and in vivo interaction between GdDnaJ and RdRp in Giardia trophozoites were confirmed by Duolink PLA. Further analysis revealed that KNK437, the inhibitor of GdDnaJ, can significantly reduce the replication of GLVs and the proliferation of Giardia. CONCLUSION: Taken together, our results suggested a potential role of GdDnaJ in regulating Giardia proliferation and GLV replication through interaction with GLV RdRp.

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.

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.

Estudo longitudinal da infecção por enteropatógenos em bezerros neonatos, com diarreia, sob diferentes estratégias de aleitamento / Longitudinal study of infection by enteropathogens in newborn calves with diarrhea under different feeding strategies

Foram utilizados 17 bezerros, recém nascidos, da raça Holandesa, com o objetivo de avaliar a influência do volume de sucedâneo nos principais patógenos causadores de diarreia neonatal. [...] Foram coletadas amostras de sangue dos bezerros com cinco dias de idade para dosagem da proteína total. A média da proteína total foi 6,33 e 6,21g/dL nos grupos 1 e 2 respectivamente. O grupo 2 apresentou tendência (p<0,1) de maior consumo de sucedâneo no período avaliado. A quantidade de sucedâneo oferecida aos animais não influenciou a incidência de diarreia e sua etiologia, ou seja, não foi observada diferença (p>0,05) na frequência das amostras positivas para cada agente entre os grupos. A frequência dos enteropatógenos nas amostras foi de 100 e 75 por cento para Cryptosporidium spp.; 28,5 e 43,7 por cento para Salmonella spp.; 28,5 e 15,6 por cento para patotipos de E. coli; 3,5 e 6,2 por cento para Rotavírus e 10,7 e 9,4 por cento para Giardia sp. nos grupos 1 e 2 respectivamente. Foram encontrados os sorotipos de Salmonella infantis e muenster. Os patotipos de E. coli isolados foram classificados como E. coli enterohemorrágica, enteropatogênica, enterotoxigênica e produtoras de toxinas Shiga 1 e 2. Foi observada associação entre o Cryptosporidium spp. e os patotipos de E. coli em 30 por cento das amostras do grupo 1 e Cryptosporidium spp. e Salmonella spp. em 45,5 por cento no grupo 2. Os resultados do presente trabalho demonstraram que o fornecimento de diferentes volumes de sucedâneo não apresentou influência sobre a incidência e etiologia da diarreia neonatal. A avaliação longitudinal dos enteropatógenos durante o período de patência da diarreia demonstrou que a associação entre eles ocorre a partir do primeiro dia da doença e destacou a importância da infecção pelo Cryptosporidium spp. agente encontrado em todos os momentos e animais.

Seventeen Holstein newborn calves were used with the objective of evaluating the influence of milk replacer volume in the pattern of pathogens causing neonatal diarrhea. […] Were collected blood samples from calves with five days of age for determination of total protein. The average total protein was 6.33 and 6.21g/dL in groups 1 and 2, respectively. The group 2 tended (p<0.1) higher consumption of milk replacer during the study period. The volume of milk replacer did not influenced the incidence of diarrhea and the frequency of positive samples for each etiologic agent between the two groups (p>0.05). Also, there was no difference (p>0.05) on the pattern of the frequency of positive samples for evaluated pathogens. The frequency of pathogens in the samples was 100 and 75 percent for Cryptosporidium, 28.5 and 43.7 percent for Salmonella spp., 28.5 and 15.6 percent for E. coli pathotypes, 3.5 and 6.2 percent for Rotavirus and 10.7 and 9.4 percent for Giardia in groups 1 and 2, respectively. Serotypes of Salmonella infantis and muenster were found. The isolated pathotypes of E. coli isolates were classified as Escherichia coli enteropathogenic, enterotoxigenic and Shiga-toxin-producing 1 and 2. Associations between Cryptosporidium spp. and E. coli pathotypes, and between Cryptosporidium spp. and Salmonella spp. were found in 30 percent of the samples in group 1 and in 45.5 percent in group 2, respectively. Our results showed that the different volumes of milk replacer did not influence the incidence and etiology of neonatal diahrrea. Longitudinal evaluation of enteropathogens during patency demonstrated that the association between the etiologic agents starts from the first day of disease. This result highlighted the great importance of the infection by Cryptosporidium spp. which was present in every moments and animals evaluated.

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.

Genetic characterization of three Cuban Trichomonas vaginalis virus. Phylogeny of Totiviridae family.

Trichomonas vaginalis can be infected with double stranded RNA (dsRNA) viruses known as T. vaginalis virus (TVV). This viral infection may have important implications for trichomonal virulence and disease pathogenesis. In this study we identified and genetic characterized three strains of TVVs isolated from T. vaginalis in Cuba. The three new predicted sequences of capsid protein and RNA-dependent RNA polymerase amounted to the previously determined 20 TVV sequences and other 21 viruses of Totiviridae family were used for a phylogenetic analysis. Four distinct monophyletic clades are shown in a phylogenetic tree. One corresponds with TVVs, other with Victorivirus, Leishmaniavirus and Eimeria brunetti virus and, other with viruses of the genus Totivirus and the last with Giardiavirus. The E. brunetti virus is identified in the phylogenetic tree as independent taxon between Leishmaniavirus and Victorivirus isolates, most closely related to Victorivirus. TVV constitute a monophyletic cluster distinguishable from all other viruses in Totiviridae family. This result suggested that TVV may be grouped in a separated genus and not inside of Giardiavirus. TVVs appear to be more closely related to protozoan viruses in the genus Leishmaniavirus and to fungal viruses in the genus Victorivirus than to other protozoan and fungal viruses in Giardiavirus and Totivirus. Among TVVs, four main groups can be recognized within Trichomonasvirus cluster, which correspond with the previous species classification proposed. Further studies, with more TVV strains, especially TVV3 and 4 strains, are needed in order to determine the phylogenetic relationship among Trichomonasvirus genus and specifically if TVV2 and 3 each also constitute a well-delimited group.

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.

A La autoantigen homologue is required for the internal ribosome entry site mediated translation of giardiavirus.

Translation of Giardiavirus (GLV) mRNA is initiated at an internal ribosome entry site (IRES) in the viral transcript. The IRES localizes to a downstream portion of 5' untranslated region (UTR) and a part of the early downstream coding region of the transcript. Recent studies indicated that the IRES does not require a pre-initiation complex to initiate translation but may directly recruit the small ribosome subunit with the help of a number of trans-activating protein factors. A La autoantigen homologue in the viral host Giardia lamblia, GlLa, was proposed as one of the potential trans-activating factors based on its specific binding to GLV-IRES in vitro. In this study, we further elucidated the functional role of GlLa in GLV-IRES mediated translation in Giardia by knocking down GlLa with antisense morpholino oligo, which resulted in a reduction of GLV-IRES activity by 40%. An over-expression of GlLa in Giardia moderately stimulated GLV-IRES activity by 20%. A yeast inhibitory RNA (IRNA), known to bind mammalian and yeast La autoantigen and inhibit Poliovirus and Hepatitis C virus IRES activities in vitro and in vivo, was also found to bind to GlLa protein in vitro and inhibited GLV-IRES function in vivo. The C-terminal domain of La autoantigen interferes with the dimerization of La and inhibits its function. An over-expression of the C-terminal domain (200-348aa) of GlLa in Giardia showed a dominant-negative effect on GLV-IRES activity, suggesting a potential inhibition of GlLa dimerization. HA tagged GlLa protein was detected mainly in the cytoplasm of Giardia, thus supporting a primary role of GlLa in translation initiation in Giardiavirus.

[Construction of GCV-specific hammerhead ribozyme recombinant vector of pyruvate kinase in Giardia lamblia].

OBJECTIVE: To construct a recombinant vector of Giardia canis virus (GCV)-specific hammerhead ribozyme of pyruvate kinase (PK) in Giardia lamblia. METHODS: Using RNA draw software, the secondary structure of PK gene in Giardia was predicted and the cleavage site of ribozyme was selected. The antisense-specific hammerhead ribozyme (PKH) targeting this site was designed. The DNA encoding the ribozyme was synthesized and the recombinant vector pGCV-PKH was constructed. The extracellular and intracellular cleavage of PK mRNA was carried out with the linear recombinant vector. Trophozoites in each group were observed with fluorescent microscopy at 24th hour after transfection. Real-time PCR was used for relative quantitative analysis of cleavage products. RESULTS: The recombinant vector of GCV-specific hammerhead ribozyme of pyruvate kinase in Giardia lamblia (pGCV-PKH) was constructed. Intracellular cleavage assays showed that the green fluorescence could only be seen in pGCV-GFP transfected trophozoites. The relative content of PK mRNA in pGCV-PKH transfected group was 33.14% of that in normal control group. It could cleave PK mRNA extracellularly and the efficiency was 58.5%. CONCLUSION: The recombinant vector can transfect Giardia trophozoites, and cleave mRNA of PK intracellularly and extracellularly.

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.

Giardiavirus internal ribosome entry site has an apparently unique mechanism of initiating translation.

Giardiavirus (GLV) utilizes an internal ribosome entry site (IRES) for translation initiation in the early branching eukaryote Giardia lamblia. Unlike most of the viral IRESs among higher eukaryotes, which localize primarily within the 5'-untranslated region (UTR), the GLV IRES comprises 253 nts of 5'UTR and the initial 264 nts in the open-reading-frame (ORF). To test if GLV IRES also functions in higher eukaryotic systems, we examined it in rabbit reticulocyte lysate (RRL) and found that it functions much less efficiently than the IRES from the Encephalomyocarditis virus (EMCV) or Cricket paralysis virus (CrPV). In contrast, both EMCV-IRES and CrPV-IRESs were inactive in transfected Giardia cells. Structure-function analysis indicated that only the stem-loop U5 from the 5'UTR and the stem-loop I plus the downstream box (Dbox) from the ORF of GLV IRES are required for limited IRES function in RRL. Edeine, a translation initiation inhibitor, did not significantly affect the function of GLV IRES in either RRL or Giardia, indicating that a pre-initiation complex is not required for GLV IRES-mediated translation initiation. However, the small ribosomal subunit purified from Giardia did not bind to GLV IRES, indicating that additional protein factors may be necessary. A member of the helicase family IBP1 and two known viral IRES binding proteins La autoantigen and SRp20 have been identified in Giardia that bind to GLV IRES in vitro. These three proteins could be involved in facilitating small ribosome recruitment for initiating translation.

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.

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.

Stable expression of green fluorescent protein mediated by GCV in Giardia canis.

Giardia canis can be infected with a double-stranded RNA virus, that is giardiavirus (G. canis virus, GCV). In this study, green fluorescent protein (GFP) was stably expressed in G. canis mediated by GCV. The plasmid pNEO/GDH/MCS/GFP, containing the neomycin phosphotransferase (NEO) encoding region flanked by the 636 nt of 5'-terminus and the 2174 nt of 3'-terminus from GCV positive strand RNA, was constructed by inserting GFP gene into downstream from the NEO gene and glutamate dehydrogenase (GDH) 5'-terminus uncoding regions on a single plasmid, and its in vitro transcript was introduced into GCV-infected G. canis by electroporation. The transfectants expressed GFP persistently under G418 selection. This stable transfection system should provide a valuable tool for genetic study of G. canis.

[The cleavage activity of GCV transfer vector-mediated hammerhead ribozyme for KRR1 in vitro transcript].

OBJECTIVE: To detect the cleavage activity of Giardia canis virus (GCV) transfer vector-mediated hammerhead ribozyme for KRR1 in vitro transcript. METHODS: Giardia, a most primitive eukaryote, has KRR1 protein responsible for ribosome biosynthesis. cDNA encoding hammerhead ribozyme flanked with various lengths of antisense RNA was cloned into a viral vector pGCV634/GFP/GCV2174 derived from the genome of GCV, KRzS flanked with 21 nt KRR1 antisense RNA on each arm, or KRzL flanked with 288 nt and 507 nt KRR1 antisense RNA. At the same time, two control groups were established: PKR without the inserted ribozyme, and TRzL flanked with 324 nt and 380 nt triosephosphate isomerase (Tim) antisense RNA. The cleavage activity of GCV transfer vector-mediated hammerhead ribozyme for KRR1 in vitro transcript was then analyzed by absolute real-time quantitative RT-PCR. RESULTS: The in vitro cleavage activities on KRR1 mRNA of the two ribozyme KRzS or KRzL were 74.0% and 81.1% respectively by the absolute real-time quantitative RT-PCR. The two control groups, PKR or TRzL, showed no effect on KRR1 mRNA in vitro. CONCLUSION: The GCV transfer vector-mediated hammerhead ribozyme shows a high cleavage activity for KRR1 in vitro transcript, which demonstrates the feasibility of using a viral vector to express a ribozyme targeted at a specific mRNA in Giardia to reduce the expression of a specific gene.

[Giardia canis virus transfection vector-mediated expression of green fluorescent protein in the parasite].

OBJECTIVE: To construct Giardia canis virus (GCV) transfection vector. METHODS: According to transcriptional start site, replication origin and packaging site of GCV genome (DQ238861), a system was developed for the expression of a foreign gene in this organism by flanking the green fluorescent protein (GFP) gene with the fragments of GCV positive-strand RNA. The transcript of the construct was synthesized in vitro with T7 RNA polymerase and used to transfect GCV-infected trophozoites by electroporation. RESULTS: The recombinant plasmid pGCV634/GFP/GCV2174 was constructed. The expression of green fluorescent protein mediated by GCV transfection vector in Giardia canis peaked at 1 d after electroporation (A490=1.8), and slowly decreased until 14 d post-transfection. CONCLUSION: The engineered GCV vector can be successfully used to introduce and efficiently express a heterologous gene in the eukaryotic microorganism.

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.

Inhibition of krr1 gene expression in Giardia canis by a virus-mediated hammerhead ribozyme.

Giardia, a most primitive eukaryote, infects several species including human and it is a major agent of waterborne outbreak of diarrhea. It has been difficult to employ standard genetic methods in the study of Giardia, but the RNA virus-based transfection system has been developed and used for the genetic manipulation. KRR1 protein is responsible for ribosome biosynthesis in Giardia. In this study, cDNA encoding hammerhead ribozyme flanked with various lengths of antisense Krr1 RNA were cloned into a viral vector pGCV634/GFP/GCV2174 derived from the genome of Giardia canis virus (GCV). RNA transcripts of the plasmids showed high cleavage activities on Krr1 mRNA in vitro. They were electroporated into GCV-infected G. canis trophozoites and Krr1 mRNA level was decreased by 72% with the ribozyme KRzS and 86% with the ribozyme KRzL, while the control ribozyme TRzS showed no effect on the level of Krr1 mRNA. The two hammerhead ribozyme transfected cells grew slowly, their internal structures got blurred and the cells were deformed. These results indicated that GCV could be useful tool for gene manipulation of G. canis.

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.