scholarly journals Potent protease inhibitors of deadly lagoviruses: rabbit hemorrhagic disease virus and European brown hare syndrome virus

2022 ◽  
Author(s):  
Krishani D Perera ◽  
David K Johnson ◽  
Scott Lovell ◽  
William Groutas ◽  
Kyeong-Ok Chang ◽  
...  
Keyword(s):  
Protease Inhibitors ◽  
Homology Modelling ◽  
Rabbit Hemorrhagic Disease Virus ◽  
Hemorrhagic Disease ◽  
Brown Hare ◽  
European Brown Hare ◽  
Rabbit Hemorrhagic Disease ◽  
The Us ◽  
European Brown Hare Syndrome

Rabbit hemorrhagic disease (RHD) and European brown hare syndrome (EBHS) are highly contagious diseases caused by lagoviruses in the Caliciviridae family and mainly affect rabbits and hares, respectively. These infectious diseases are associated with high mortality and a serious threat to domesticated (farmed and pet) and wild rabbits and hares, including endangered species such as Riparian brush rabbits. In the US, only isolated cases of RHD had been reported until Spring 2020. However, RHD caused by RHD type 2 virus (RHDV2) was unexpectedly reported in April 2020 in New Mexico and has subsequently spread to several US states infecting wild rabbits and hares. Since it is almost impossible to control and eradicate the virus from wild animals, it is highly likely RHD will become endemic in the US. Vaccines are available for RHD, however, there is no specific treatment for these deadly diseases. RHDV and EBHSV encode a 3C-like protease (3CLpro), which is essential for virus replication and a promising target for antiviral drug development. We have previously generated focused small molecule libraries of 3CLpro inhibitors and demonstrated the in vitro potency and in vivo efficacy of some protease inhibitors against viruses that encode 3CLpro including caliciviruses and coronaviruses. Here we established the enzyme assay and cell-based assays for these uncultivable viruses to determine the in vitro activity of 3CLpro inhibitors, including GC376, a protease inhibitor being developed for feline infectious peritonitis, and identified potent inhibitors of RHDV1 and 2 and EBHSV. In addition, structure-activity relationship study and homology modelling of the 3CLpros and inhibitors revealed that lagoviruses share similar structural requirements for 3CLpro inhibition with other caliciviruses.

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 Proteolytic Processing of Sapovirus ORF1 Polyprotein

2005 ◽  
Vol 79 (12) ◽  
pp. 7283-7290 ◽  
Author(s):  
Tomoichiro Oka ◽  
Kazuhiko Katayama ◽  
Satoko Ogawa ◽  
Grant S. Hansman ◽  
Tsutomu Kageyama ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proteolytic Processing ◽  
Open Reading Frames ◽  
Rabbit Hemorrhagic Disease Virus ◽  
Translation System ◽  
Hemorrhagic Disease ◽  
Rabbit Hemorrhagic Disease ◽  
Capsid Protein Vp1

ABSTRACT The genome of Sapovirus (SaV), a causative agent of gastroenteritis in humans and swine, contains either two or three open reading frames (ORFs). Functional motifs characteristic to the 2C-like NTPase (NTPase), VPg, 3C-like protease (Pro), 3D-like RNA-dependent RNA polymerase (Pol), and capsid protein (VP1) are encoded in the ORF1 polyprotein, which is afterwards cleaved into the nonstructural and structural proteins. We recently determined the complete genome sequence of a novel human SaV strain, Mc10, which has two ORFs. To investigate the proteolytic cleavage of SaV ORF1 and the function of protease on the cleavage, both full-length and truncated forms of the ORF1 polyprotein either with or without mutation in 1171Cys to Ala of the GDCG motif were expressed in an in vitro coupled transcription-translation system. The translation products were analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by immunoprecipitation with region-specific antibodies. The ORF1 polyprotein was processed into at least 10 major proteins: p11, p28, p35, p32, p14, p70, p60, p66, p46, and p120. Seven of these products were arranged in the following order: NH2-p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)-COOH. p66, p46 and p120 were precursors of p28-p35 (NTPase), p32-p14 (VPg), and p32-p14 (VPg)-p70 (Pro-Pol), respectively. Mutagenesis in the 3C-like protease motif fully abolished the proteolytic activity. The cleavage map of SaV ORF1 is similar to those of other heretofore known members of the family Caliciviridae, especially to rabbit hemorrhagic disease virus, a member of the genus Lagovirus.

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 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 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.

Development of a RT-PCR for the Detection of European Brown Hare Syndrome Virus

2011 ◽  
Vol 271-273 ◽  
pp. 410-416 ◽  
Author(s):  
Yang Ze Xiao ◽  
Li Rui Li ◽  
Yin Wang ◽  
Xue Ping Yao ◽  
Xue Qing Han ◽  
...  
Keyword(s):  
Pasteurella Multocida ◽  
Brown Hare ◽  
Rt Pcr ◽  
European Brown Hare ◽  
Vp60 Gene ◽  
Specificity And Sensitivity ◽  
Overlap Extension ◽  
Pcr Method ◽  
European Brown Hare Syndrome

To develop the reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of European brown hare syndrome virus (EBHSV), 3 specific primers and 9 overlapping oligo primers were designed to amplify a fragment of 335 nucleotides from the genome of EBHSV according to the genome sequences published in GenBank, and a 359 bp DNA fragment of the capsid protein(VP60)gene of EBHSV was synthesized in vitro by using overlap extension PCR to construct the recombinant plasmids pGM-T-EBHSV, then the RT-PCR method was described through the optimization of reaction conditions, sensitivity and specificity tests. Results showed the RT-PCR method was supplied for the detection of EBHSV with good specificity and sensitivity. The sensitivity of the RT-PCR could reach about 50 copies of cloned viral genomic fragments (pGM-T-EBHSV), and no amplifications for RHDV, Pasteurella multocida, E.coliandSalmonellafrom rabbits detection by this approach.

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