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C.c. Wang - One of the best experts on this subject based on the ideXlab platform.
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Protein synthesis in Giardia lamblia may involve interaction between a downstream box (DB) in mRNA and an anti-DB in the 16S-like ribosomal RNA.
Molecular and Biochemical Parasitology, 1998Co-Authors: A.l. Wang, C W Botka, C.c. WangAbstract:Giardia lamblia, a parasitic protozoan, has been regarded as one of the most conserved eukaryotes evolved from the prokaryotes. One of its unique features appears to be the unusually short 5'-untranslated regions (UTR) (1-6 nucleotides (nts)) and the apparent absence of 5'-cap structures from its mRNAs. Transfection of the Giardia trophozoites with luciferase-encoding chimeric transcripts, flanked by the 5'- and 3'-ends of Giardiavirus (GLV) (+)-strand RNA, indicated that the translational efficiency was enhanced by 5000-fold when the 5'-viral sequence extended 264 nts into the capsid coding region and fused with the luciferase open reading frame (ORF). A 13-nt downstream box (DB) was identified within this region which complements a 15-nt sequence between nts # 1382 and 1396 near the 3'-end of the Giardia 16S-like ribosomal RNA (the anti-DB). Deletion or scrambling of this DB in the mRNA leads to a significant loss of the translational efficiency in Giardia. A Shine-Dalgarno (SD)-like element was also identified at 9-14 nts upstream from the initiation codon in the viral (+)-strand RNA, but alteration of its sequence led to no change in translation. Using the sequence complementary to ribosomal anti-DB to probe the Giardia mRNAs available in the databases, each mRNA was found to contain a putative DB with an average length from 8 to 13 nts. It is thus possible that initiation of translation in Giardia may involve a DB in the coding region of mRNA that may bind to a putative anti-DB in the small ribosomal RNA through base pairing. This mechanism of ribosome recruitment, which finds a potential parallel in Escherichia coli, could illustrate a relatively close distance between Giardia and prokaryotes in terms of translation initiation, and may provide a model for studying the evolution of translation machinery.
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by Giardiavirus in Giardia lamblia
1996Co-Authors: A.l. Wang, C.c. Wang, Alice L. Wang, C. WangAbstract:foreign gene by Giardiavirus in Giardia lamblia. Amplification, expression, and packaging of
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Amplification, expression, and packaging of a foreign gene by Giardiavirus in Giardia lamblia.
Journal of virology, 1996Co-Authors: A.l. Wang, C.c. WangAbstract: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.
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Identification of cis-acting signals in the Giardiavirus (GLV) genome required for expression of firefly luciferase in Giardia lamblia.
RNA (New York N.Y.), 1996Co-Authors: C.c. WangAbstract:Giardiavirus (GLV) is a 6,277-bp double-stranded RNA virus of Giardia lamblia, one of the earliest eukaryotic divergents from the prokaryotes. Our previous success in GLV-mediated transfection of G. lamblia has provided an effective way of monitoring the mechanisms underlining GLV gene replication and mRNA translation in this organism. Here we have investigated the cis-acting signals in the GLV genome that regulate replication, transcription, and translation of an inserted firefly luciferase gene in GLV-infected G. lamblia. By modifying the two terminal regions of a full-length GLV cDNA clone used to flank a luciferase gene, various in vitro chimeric transcripts were generated and introduced into GLV-infected G. lamblia via electroporation. Expression of luciferase (+) strand and (-) strand RNAs in the transfected cells was monitored and the luciferase activity assayed. The results indicated that the 5'-untranslated region (UTR) of 366 nt and the 3'-terminal 2,022 nt of the viral transcript are both needed for optimal expression of the two RNA strands. Although the entire 5'-UTR is needed for the chimeric mRNA synthesis, both the primary sequence and the secondary structure at the 3' end of GLV transcript are essential for the synthesis of (-) strand RNA. When the 5' end of GLV transcript was extended 265 nt into the capsid protein open reading frame and fused with that of luciferase, there was no change in the level of luciferase chimeric RNA, but a 5,000-fold increase of luciferase activity was observed that may be attributed to an enhanced translational efficiency of the chimeric mRNA in G. lamblia.
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Stable coexpression of a drug-resistance gene and a heterologous gene in an ancient parasitic protozoan Giardia lamblia
Molecular and Biochemical Parasitology, 1996Co-Authors: Alice L. Wang, C.c. WangAbstract:Abstract Manipulation of gene expression in Giardia lamblia , one of the most ancient eukaryotes, may provide insights into the evolutionary transition from prokaryotes to eukaryotes. Two recent successes in transient expression of the firefly luciferase ( luc ) gene in G. lamblia were mediated by a 5′-untranslated region (UTR) of the Giardia glutamate dehydrogenase ( gdh ) gene [1] and a Giardiavirus (GLV) genomic transcript [2], respectively. We now report a stable coexpression of luc gene with a neomycin phosphotransferase ( neo r ) gene in G. lamblia . An in vitro transcript of the construct pC670-Neo, containing the neo r encoding region flanked with the 5′ 670 nucleotides (nt) and the 3′ 2022 nt portion of GLV positive strand RNA, was electroporated into G. lamblia trophozoites that were infected with GLV. G418-resistant Giardia trophozoites were cloned, and the neo r mRNA in these clones was found to increase with increasing G418 pressure. This drug resistance remained stable upon continuous in vitro cultivation in the absence of G418 for over 15 days. Another plasmid pNeo/GDH/Luc, was constructed by inserting luc gene downstream from the neo r gene and the 193 nt 5′ portion of gdh gene in pC670-Neo, and its bicistronic in vitro transcript was introduced into GLV-infected G. lamblia by electroporation. The transfectants demonstrated G418-resistance and persistent luciferase activity at levels parallel to the amount of G418 used for selection, peaking at a level of several thousand-fold above the background. Taken together, these data indicate that the neo r gene provides an effective selection marker for transformation of Giardia trophozoites, and the bicistronic RNA transfection vector may open the way for functional analysis of other genes in Giardia .
Ching C. Wang - One of the best experts on this subject based on the ideXlab platform.
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A La Autoantigen Homologue Is Required for the Internal Ribosome Entry Site Mediated Translation of Giardiavirus
PLoS ONE, 2011Co-Authors: Srinivas Garlapati, Ashesh A. Saraiya, Ching C. WangAbstract: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.
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2011).A la autoantigen homologue is required for the internal ribosome entry site mediated translation of Giardiavirus
2010Co-Authors: Srinivas Garlapati, Ashesh A. Saraiya, Ching C. WangAbstract: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 59 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, thu
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Giardiavirus internal ribosome entry site has an apparently unique mechanism of initiating translation.
PLoS ONE, 2009Co-Authors: Srinivas Garlapati, Ching C. WangAbstract: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.
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Giardiavirus Internal Ribosome Entry Site Has an Apparently Unique Mechanism of Initiating Translation
2009Co-Authors: Srinivas Garlapati, Ching C. WangAbstract: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 59-untranslated region (UTR), the GLV IRES comprises 253 nts of 59UTR 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 59UTR 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 involve
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Structural elements in the 5'-untranslated region of Giardiavirus transcript essential for internal ribosome entry site-mediated translation initiation.
Eukaryotic Cell, 2005Co-Authors: Srinivas Garlapati, Ching C. WangAbstract:Translation of uncapped Giardiavirus (GLV) mRNA in Giardia lamblia requires the presence of a 5′-untranslated region (5′-UTR) and a viral capsid coding region. We used dicistronic viral constructs to show that the downstream 253 nucleotides (nt) of the 5′-UTR plus the initial 264-nt capsid coding region constitute an internal ribosome entry site (IRES). Predicted secondary structures in the 253-nt 5′-UTR include stem-loops U3, U4a, U4b, U4c, and U5. Chemical and enzymatic probing analysis confirmed the presence of all predicted stem-loops except U4a. Disruption of stem-loop structures U3 and U5 by site-directed mutagenesis resulted in a drastic reduction in translation of a monocistronic viral transcript, which could be restored by compensatory sequence changes. Mutations disrupting stem-loops U4b and U4c do not exert an appreciable effect on translation, but certain sequences in the U4a region and in U4b do appear to play important roles in the IRES. Structural analysis also suggests that an 8-nt U3 loop sequence (nt 147 to 154) pairs with an 8-nt downstream sequence (nt 168 to 175) to form a pseudoknot. Disruption of this pseudoknot by mutagenesis resulted in a drastic reduction in translation, which could be restored by compensatory sequence changes. This study has defined the secondary structure in the 5′-UTR of the IRES. Together with the previous results, we have now completed analysis of the entire structure of GLV IRES and fully defined the functionally essential structural elements in it.
A.l. Wang - One of the best experts on this subject based on the ideXlab platform.
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Protein synthesis in Giardia lamblia may involve interaction between a downstream box (DB) in mRNA and an anti-DB in the 16S-like ribosomal RNA.
Molecular and Biochemical Parasitology, 1998Co-Authors: A.l. Wang, C W Botka, C.c. WangAbstract:Giardia lamblia, a parasitic protozoan, has been regarded as one of the most conserved eukaryotes evolved from the prokaryotes. One of its unique features appears to be the unusually short 5'-untranslated regions (UTR) (1-6 nucleotides (nts)) and the apparent absence of 5'-cap structures from its mRNAs. Transfection of the Giardia trophozoites with luciferase-encoding chimeric transcripts, flanked by the 5'- and 3'-ends of Giardiavirus (GLV) (+)-strand RNA, indicated that the translational efficiency was enhanced by 5000-fold when the 5'-viral sequence extended 264 nts into the capsid coding region and fused with the luciferase open reading frame (ORF). A 13-nt downstream box (DB) was identified within this region which complements a 15-nt sequence between nts # 1382 and 1396 near the 3'-end of the Giardia 16S-like ribosomal RNA (the anti-DB). Deletion or scrambling of this DB in the mRNA leads to a significant loss of the translational efficiency in Giardia. A Shine-Dalgarno (SD)-like element was also identified at 9-14 nts upstream from the initiation codon in the viral (+)-strand RNA, but alteration of its sequence led to no change in translation. Using the sequence complementary to ribosomal anti-DB to probe the Giardia mRNAs available in the databases, each mRNA was found to contain a putative DB with an average length from 8 to 13 nts. It is thus possible that initiation of translation in Giardia may involve a DB in the coding region of mRNA that may bind to a putative anti-DB in the small ribosomal RNA through base pairing. This mechanism of ribosome recruitment, which finds a potential parallel in Escherichia coli, could illustrate a relatively close distance between Giardia and prokaryotes in terms of translation initiation, and may provide a model for studying the evolution of translation machinery.
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by Giardiavirus in Giardia lamblia
1996Co-Authors: A.l. Wang, C.c. Wang, Alice L. Wang, C. WangAbstract:foreign gene by Giardiavirus in Giardia lamblia. Amplification, expression, and packaging of
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Amplification, expression, and packaging of a foreign gene by Giardiavirus in Giardia lamblia.
Journal of virology, 1996Co-Authors: A.l. Wang, C.c. WangAbstract: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.
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Virus-mediated expression of firefly luciferase in the parasitic protozoan Giardia lamblia.
Molecular and Cellular Biology, 1995Co-Authors: A.l. Wang, Ching C. WangAbstract: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.
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Sequences at four termini of the Giardiavirus double-stranded RNA.
Gene, 1995Co-Authors: C.c. Wang, Hungmin Yang, A.l. WangAbstract: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.
Nobuhiro Suzuki - One of the best experts on this subject based on the ideXlab platform.
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Novel Victorivirus from a Pakistani Isolate of Alternaria alternata Lacking a Typical Translational Stop/Restart Sequence Signature
Viruses, 2019Co-Authors: Atif Jamal, Yukiyo Sato, Sabitree Shahi, Wajeeha Shamsi, Hideki Kondo, Nobuhiro SuzukiAbstract:The family Totiviridae currently contains five genera Totivirus, Victorivirus, Leishmavirus, Trichomonasvirus, and Giardiavirus. Members in this family generally have a set of two-open reading frame (ORF) elements in their genome with the 5′-proximal ORF (ORF1) encoding a capsid protein (CP) and the 3′-proximal one (ORF2) for RNA-dependent RNA polymerase (RdRp). How the downstream open reading frames (ORFs) are expressed is genus-specific. All victoriviruses characterized thus far appear to use the stop/restart translation mechanism, allowing for the expression of two separate protein products from bicitronic genome-sized viral mRNA, while the totiviruses use a −1 ribosomal frame-shifting that leads to a fusion product of CP and RdRp. We report the biological and molecular characterization of a novel victorivirus termed Alternaria alternata victorivirus 1 (AalVV1) isolated from Alternaria alternata in Pakistan. The phylogenetic and molecular analyses showed AalVV1 to be distinct from previously reported victoriviruses. AalVV1 appears to have a sequence signature required for the −1 frame-shifting at the ORF1/2 junction region, rather than a stop/restart key mediator. By contrast, SDS–polyacrylamide gel electrophoresis and peptide mass fingerprinting analyses of purified virion preparations suggested the expression of two protein products, not a CP-RdRp fusion product. How these proteins are expressed is discussed in this study. Possible effects of infection by this virus were tested in two fungal species: A. alternata and RNA silencing proficient and deficient strains of Cryphonectria parasitica, a model filamentous fungus. AalVV1 showed symptomless infection in all of these fungal strains, even in the RNA silencing deficient C. parasitica strain.
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novel victorivirus from a pakistani isolate of alternaria alternata lacking a typical translational stop restart sequence signature
Viruses, 2019Co-Authors: Atif Jamal, Yukiyo Sato, Sabitree Shahi, Wajeeha Shamsi, Hideki Kondo, Nobuhiro SuzukiAbstract:The family Totiviridae currently contains five genera Totivirus, Victorivirus, Leishmavirus, Trichomonasvirus, and Giardiavirus. Members in this family generally have a set of two-open reading frame (ORF) elements in their genome with the 5′-proximal ORF (ORF1) encoding a capsid protein (CP) and the 3′-proximal one (ORF2) for RNA-dependent RNA polymerase (RdRp). How the downstream open reading frames (ORFs) are expressed is genus-specific. All victoriviruses characterized thus far appear to use the stop/restart translation mechanism, allowing for the expression of two separate protein products from bicitronic genome-sized viral mRNA, while the totiviruses use a −1 ribosomal frame-shifting that leads to a fusion product of CP and RdRp. We report the biological and molecular characterization of a novel victorivirus termed Alternaria alternata victorivirus 1 (AalVV1) isolated from Alternaria alternata in Pakistan. The phylogenetic and molecular analyses showed AalVV1 to be distinct from previously reported victoriviruses. AalVV1 appears to have a sequence signature required for the −1 frame-shifting at the ORF1/2 junction region, rather than a stop/restart key mediator. By contrast, SDS–polyacrylamide gel electrophoresis and peptide mass fingerprinting analyses of purified virion preparations suggested the expression of two protein products, not a CP-RdRp fusion product. How these proteins are expressed is discussed in this study. Possible effects of infection by this virus were tested in two fungal species: A. alternata and RNA silencing proficient and deficient strains of Cryphonectria parasitica, a model filamentous fungus. AalVV1 showed symptomless infection in all of these fungal strains, even in the RNA silencing deficient C. parasitica strain.
Leigh Owens - One of the best experts on this subject based on the ideXlab platform.
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Natural infection of the redclaw crayfish Cherax quadricarinatus with presumptive spawner-isolated mortality virus.
Diseases of Aquatic Organisms, 2000Co-Authors: Leigh Owens, Catriona McelneaAbstract:Crayfish farmers reported reduced tolerance of stress in specimens of Cherax quadricarinatus, which were formerly robust crayfish. Furthermore, one farmer reported a large reduction in yield with final harvest only equaling the stocking weight. Upon trapping, one-third of the crayfish regularly died overnight and a further one-third died on the sorting tray during sexing of juveniles (~3 mo old). Histopathological examination revealed very light (1 or 2 cells per section) infections with Cherax Giardiavirus and sometimes mild atrophy of hepatopancreatic cells. Gene probe analysis with a DIG-labeled spawner-isolated mortality virus (SMV) probe demonstrated extensive positive signals in nuclei of many tissues. The hepatopancreas, the midgut, glands associated with the midgut, the epithelium of seminal ducts and follicle cells surrounding oocytes gave the strongest positive signals. Nuclei of the heart, haemocytes, connective tissue and subcutis gave positive signals in some individuals. Although signals were intense and extensive, cytolysis of infected cells was very limited. The possibility of cross infections of SMV between prawns and freshwater crayfish is of international quarantine significance.
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Histopathological surveys of the redclaw freshwater crayfish, Cherax quadricarinatus, in Australia
Aquaculture, 1999Co-Authors: Brett F. Edgerton, Leigh OwensAbstract:Histopathological surveys on Cherax quadricarinatus from farms in northern Queensland were conducted in 1992 and 1996. C. quadricarinatus bacilliform virus (CqBV) and C. Giardiavirus-like virus were highly prevalent in both surveys. The prevalence of CqBV in three farms was markedly higher in the 1996 survey than in the 1992 survey. However increased mortalities were not reported by farmers in this period. Bacteraemia and Psorospermium sp. were common in both surveys. Enteric bacterial infections were common at one farm in 1996. Microsporidiosis was observed in two crayfish from one farm in 1992. Haemocytic enteritis was common in two farms in 1996, and black spots were observed on crayfish from one farm in 1992, and from two different farms in 1996. Several other rare idiopathic lesions were observed. The need for more thorough data sets on the prevalence and incidence of disease causing organisms in freshwater crayfish is highlighted.
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Histopathological surveys of the redclaw freshwater crayfish, Cherax quadricarinatus, in Australia
Aquaculture, 1999Co-Authors: Brett F. Edgerton, Leigh OwensAbstract:Histopathological surveys on Cherax quadricarinatus from farms in northern Queensland were conducted in 1992 and 1996. C. quadricarinatus bacilliform virus (CqBV) and C. Giardiavirus-like virus were highly prevalent in both surveys. The prevalence of CqBV in three farms was markedly higher in the 1996 survey than in the 1992 survey. However increased mortalities were not reported by farmers in this period. Bacteraemia and Psorospermium sp. were common in both surveys. Enteric bacterial infections were common at one farm in 1996. Microsporidiosis was observed in two crayfish from one farm in 1992. Haemocytic enteritis was common in two farms in 1996, and black spots were observed on crayfish from one farm in 1992, and from two different farms in 1996. Several other rare idiopathic lesions were observed. The need for more thorough data sets on the prevalence and incidence of disease causing organisms in freshwater crayfish is highlighted.
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Age at first infection of Cherax quadricarinatus by Cherax quadricarinatus bacilliform virus and Cherax Giardiavirus-like virus, and production of putative virus-free crayfish
Aquaculture, 1997Co-Authors: Brett F. Edgerton, Leigh OwensAbstract:Abstract This study determined the age at which Cherax quadricarinatus is first susceptible to infection by Cherax quadricarinatus bacilliform virus (CqBV) and Cherax Giardiavirus -like virus (CGV). The first CqBV infected juvenile was diagnosed 2 weeks after the juvenile moulted into stage 3 (week 2), and the prevalence of CqBV rose steadily to 17.1% at the completion of the experiment (week 7). CGV was first diagnosed at week 1 and its prevalence rose dramatically to reach 58% by week 3 and 86% by week 6. Presumptive virus-free juvenile C. quadricarinatus were produced from infected females by removing the eggs at the eyed stage, sterilising and hatching them, and culturing the juveniles in uncontaminated water. These procedures may be used to prevent CqBV and CGV infections in aquaculture, or more importantly, prevent translocations of these viruses.