scholarly journals Expression of Enzymatically Active Rabbit Hemorrhagic Disease Virus RNA-Dependent RNA Polymerase in Escherichia coli

1998 ◽  
Vol 72 (4) ◽  
pp. 2999-3004 ◽  
Author(s):  
Ana López Vázquez ◽  
José M. Martín Alonso ◽  
Rosa Casais ◽  
José A. Boga ◽  
Francisco Parra
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Disease Virus ◽  
Rabbit Hemorrhagic Disease Virus ◽  
Hemorrhagic Disease ◽  
Coding Region ◽  
Recombinant Polypeptide ◽  
Rna Dependent Rna Polymerase ◽  
Rabbit Hemorrhagic Disease

ABSTRACT The rabbit hemorrhagic disease virus (RHDV) (isolate AST/89) RNA-dependent RNA-polymerase (3Dpol) coding region was expressed in Escherichia coli by using a glutathioneS-transferase-based vector, which allowed milligram purification of a homogeneous enzyme with an expected molecular mass of about 58 kDa. The recombinant polypeptide exhibited rifampin- and actinomycin D-resistant, poly(A)-dependent poly(U) polymerase. The enzyme also showed RNA polymerase activity in in vitro reactions with synthetic RHDV subgenomic RNA in the presence or absence of an oligo(U) primer. Template-size products were synthesized in the oligo(U)-primed reactions, whereas in the absence of added primer, RNA products up to twice the length of the template were made. The double-length RNA products were double stranded and hybridized to both positive- and negative-sense probes.

scholarly journals Mutation Analysis of the GDD Sequence Motif of a Calicivirus RNA-Dependent RNA Polymerase

2000 ◽  
Vol 74 (8) ◽  
pp. 3888-3891 ◽  
Author(s):  
Ana López Vázquez ◽  
José M. Martín Alonso ◽  
Francisco Parra
Keyword(s):  
Rna Polymerase ◽  
Metal Ion ◽  
Polymerase Activity ◽  
Rabbit Hemorrhagic Disease Virus ◽  
Sequence Motif ◽  
Hemorrhagic Disease ◽  
Rna Dependent Rna Polymerase ◽  
Rabbit Hemorrhagic Disease ◽  
Aspartate Residue

ABSTRACT The RNA-dependent RNA polymerase from rabbit hemorrhagic disease virus, a calicivirus, is known to have a conserved GDD amino acid motif and several additional regions of sequence homology with all types of polymerases. To test whether both aspartic acid residues are in fact involved in the catalytic activity and metal ion coordination of the enzyme, several defined mutations have been made in order to replace them by glutamate, asparagine, or glycine. All six mutant enzymes were produced in Escherichia coli, and their in vitro poly(U) polymerase activity was characterized. The results demonstrated that the first aspartate residue was absolutely required for enzyme function and that some flexibility existed with respect to the second, which could be replaced by glutamate.

scholarly journals A Review on the Methods Used for the Detection and Diagnosis of Rabbit Hemorrhagic Disease Virus (RHDV)

2021 ◽  
Vol 9 (5) ◽  
pp. 972
Author(s):  
Joana Abrantes ◽  
Ana M. Lopes
Keyword(s):  
Disease Virus ◽  
Diagnostic Tests ◽  
High Throughput Sequencing ◽  
Current Knowledge ◽  
European Rabbit ◽  
Rabbit Hemorrhagic Disease Virus ◽  
Hemorrhagic Disease ◽  
Rabbit Hemorrhagic Disease ◽  
Detection And Diagnosis

Since the early 1980s, the European rabbit (Oryctolagus cuniculus) has been threatened by the rabbit hemorrhagic disease (RHD). The disease is caused by a lagovirus of the family Caliciviridae, the rabbit hemorrhagic disease virus (RHDV). The need for detection, identification and further characterization of RHDV led to the development of several diagnostic tests. Owing to the lack of an appropriate cell culture system for in vitro propagation of the virus, much of the methods involved in these tests contributed to our current knowledge on RHD and RHDV and to the development of vaccines to contain the disease. Here, we provide a comprehensive review of the RHDV diagnostic tests used since the first RHD outbreak and that include molecular, histological and serological techniques, ranging from simpler tests initially used, such as the hemagglutination test, to the more recent and sophisticated high-throughput sequencing, along with an overview of their potential and their limitations.

scholarly journals ATP Binding and ATPase Activities Associated with Recombinant Rabbit Hemorrhagic Disease Virus 2C-Like Polypeptide

2000 ◽  
Vol 74 (22) ◽  
pp. 10846-10851 ◽  
Author(s):  
M. Soledad Marín ◽  
Rosa Casais ◽  
José M. Martín Alonso ◽  
Francisco Parra
Keyword(s):  
Disease Virus ◽  
Site Directed Mutagenesis ◽  
Rabbit Hemorrhagic Disease Virus ◽  
Atp Binding ◽  
Hemorrhagic Disease ◽  
Rabbit Hemorrhagic Disease ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal

ABSTRACT The carboxy-terminal region of the rabbit hemorrhagic disease virus p37 polyprotein cleavage product has been expressed inEscherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant GST-Δ2C protein showed in vitro ATP-binding and ATPase activities. Site-directed mutagenesis studies of the conserved residues G522 and T529 in motif A, D566 and E567 in motif B, and K600 in motif C were also performed. These results provide the first experimental characterization of a 2C-like ATPase activity in a member of the Caliciviridae.

scholarly journals Detection of Viral Proteins after Infection of Cultured Hepatocytes with Rabbit Hemorrhagic Disease Virus

1998 ◽  
Vol 72 (5) ◽  
pp. 4492-4497 ◽  
Author(s):  
Matthias König ◽  
Heinz-Jürgen Thiel ◽  
Gregor Meyers
Keyword(s):  
Disease Virus ◽  
Genome Organization ◽  
Gradient Centrifugation ◽  
Liver Perfusion ◽  
Viral Proteins ◽  
Rabbit Hemorrhagic Disease Virus ◽  
Hemorrhagic Disease ◽  
Rabbit Hemorrhagic Disease ◽  
Infected Cells

ABSTRACT The calicivirus rabbit hemorrhagic disease virus (RHDV), which replicates predominantly in the livers of infected rabbits, cannot be propagated in tissue culture. To enable the performance of in vitro studies, rabbit hepatocytes were isolated by liver perfusion and gradient centrifugation. After inoculation with purified RHDV, more than 50% of the cells proved to be infected. Protein analyses led to the detection of 13 RHDV-specific polypeptides within the infected cells. These proteins were assigned to defined regions of the viral genome, resulting in a refined model of RHDV genome organization.

scholarly journals Recovery of Infectious Rabbit Hemorrhagic Disease Virus from Rabbits after Direct Inoculation with In Vitro-Transcribed RNA

2006 ◽  
Vol 80 (13) ◽  
pp. 6597-6602 ◽  
Author(s):  
Guangqing Liu ◽  
Yuying Zhang ◽  
Zheng Ni ◽  
Tao Yun ◽  
Zutian Sheng ◽  
...  
Keyword(s):  
Disease Virus ◽  
Infectious Clone ◽  
Full Length ◽  
Rabbit Hemorrhagic Disease Virus ◽  
Progeny Virus ◽  
Cdna Clones ◽  
Hemorrhagic Disease ◽  
Rabbit Hemorrhagic Disease ◽  
Rna Transcripts

ABSTRACT We report the first full-length infectious clone of strain JX/CHA/97 of rabbit hemorrhagic disease virus (RHDV). The transcripts from the full-length cDNA clones were infectious when they were directly injected into rabbits. The sequence of the virus recovered from the rabbits was identical to that of the injected RNA transcripts. The cDNA clone was engineered to contain one silent nucleotide change to create an EcoRV site (A to T at nucleotide 2908). The genetic marker was retained in the recovered progeny virus. The transfection of RNA transcripts into RK-13 cells resulted in the synthesis of viral antigens, indicating that the cDNA clones were replication competent. This stable infectious molecular clone should be an important tool for developing a better understanding of the molecular biology and pathogenesis of RHDV.

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