Identification of further proteolytic cleavage sites in the Southampton calicivirus polyprotein by expression of the viral protease in E. coli.
Southampton virus (SV) is a human enteric calicivirus with a positive-sense RNA genome which encodes a protease as part of a large precursor polyprotein. Expression vectors based on pRSET were constructed carrying the entire 3C-like viral protease (3Cpro) sequence together with flanking sequences from a region of the viral genome 3'-distal to the putative helicase-encoding region. Expression from these vectors in E. coli resulted in discrete protein products with smaller than expected molecular sizes. This confirmed that an active viral protease was produced in E. coli and that the protease was capable of cleaving the expressed protein at defined sites. Expressed cleavage products surrounding the protease region of the viral polyprotein were separated by SDS-PAGE, transferred to PVDF membranes and subjected to N-terminal sequence analysis. Cleavage occurred at an EG dipeptide at the N terminus of the putative VPg (961E/GKNKG) and also at the protease/polymerase boundary (1280E/GGDKG). The N terminus of the protease was released from the VPg C terminus at an EA dipeptide in the sequence 1099E/APPTL. These studies demonstrate that SV enteric calicivirus encodes a 3C-like protease with a specificity similar to the picornaviral 3C protease and that the SV polyprotein is cleaved into at least six mature products. (+info)
Organization of the canine calicivirus genome from the RNA polymerase gene to the poly(A) tail.
In recent years a wealth of data has become available about the caliciviruses that infect humans, as well as those which infect a range of animal species, notably cats, rabbits, pigs and marine animals. However, in the two decades since the earliest reports of calicivirus infection in dogs, very little has become known about the epidemiology, pathogenicity and molecular biology of the caliciviruses that may infect canines. In 1990, a canine calicivirus (CaCV) was isolated from a 2-month-old diarrhoeic domestic dog in Japan. This virus, which can be grown in cultured cells of canine origin, has the classic 'Star of David' morphology of caliciviruses, and the one major structural protein was shown to be immunogenic in dogs. In this study, a 3.8 kb region of the genome of this CaCV isolate from the RNA polymerase gene to the 3' poly(A) tail was cloned and sequenced, and phylogenetic analysis was undertaken in order to establish the relationship of CaCV to other animal and human caliciviruses. This CaCV isolate had a nucleotide sequence, genomic organization and phylogenetic position closest to, but clearly distinct from, both feline calicivirus and San Miguel sea lion virus isolates. These findings suggest that CaCV represents a new clade of animal caliciviruses, presumably as a member of the recently proposed new genus Vesivirus. (+info)
Isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves.
Small round viruses (SRV) were isolated from the faeces of diarrhoeic calves from three farms. All three SRV preparations caused diarrhoea experimentally in gnotobiotic calves. Each preparation contained viral particles of two morphological types, "astrovirus-like" and "calicivirus-like", and from one preparation the two particle types were separated from each other. The calicivirus-like agent ("Newbury agent") was 33 nm in diameter, and caused diarrhoea in gnotobiotic calves with villous atrophy and D-xylose malabsorption. This virus did not infect cell cultures. The astrovirus-like agent did not cause diarrhoea in two gnotobiotic calves; however, it infected cell cultures (primary calf kidney) and the infected cells immunofluoresced with convalescent gnotobiotic-calf antiserum. The astrovirus-like agents in the three preparations were antigenically related. Experiments in calves showed that there was a degree of cross-protection between the three SRV preparations, as judged by the presence or absence of diarrhoea, but that at least three unrelated pathogens were present. (+info)
Genetic analysis of the RNA polymerase gene of caliciviruses from dogs and cats.
Caliciviruses that infect animals including humans cause a specific disease syndrome in their respective hosts. Feline calicivirus (FCV) is a major pathogen of respiratory disease of cats, and human caliciviru is a causative agent of diarrhea. It has been suggested, furthermore, that FCV and newly recognized canine calicivirus (CaCV) may also be possible causes of diarrhea in these animal species. In this study nucleotide sequence of the RNA polymerase gene of two caliciviruses of canine origin, namely CaCV strain No. 48 and FCV-like strain Sapporo/283, and a number of FCV strains of respiratory and enteric origins was examined. The length of sequenced region, from the 5'LKDEL motif through the 3'YGDD motif of the gene, was 555 bp for CaCV No. 48 strain and 552 bp for the other FCV strains including Sapporo/283 strain. In phylogenetic analysis, CaCV No. 48 strain grouped as a distinct branch sharing ancestral roots with San Miguel sea lion virus, and FCVs formed one compact group in which Sapporo/283 strain was included. (+info)
Molecular characterization of a porcine enteric calicivirus genetically related to Sapporo-like human caliciviruses.
Porcine enteric calicivirus (PEC) is associated with diarrhea in pigs, and to date it is the only cultivable enteric calicivirus (tissue culture-adapted [TC] PEC/Cowden). Based on sequence analysis of cDNA clones and reverse transcription-PCR products, TC PEC/Cowden has an RNA genome of 7,320 bp, excluding its 3' poly(A)(+) tail. The genome is organized in two open reading frames (ORFs), similar to the organizations of the human Sapporo-like viruses (SLVs) and the lagoviruses. ORF1 encodes the polyprotein that is fused to and contiguous with the capsid protein. ORF2 at the 3' end encodes a small basic protein of 164 amino acids. Among caliciviruses, PEC has the highest amino acid sequence identities in the putative RNA polymerase (66%), 2C helicase (49.6%), 3C-like protease (43.7%), and capsid (39%) regions with the SLVs, indicating that PEC is genetically most closely related to the SLVs. The complete RNA genome of wild-type (WT) PEC/Cowden was also sequenced. Sequence comparisons revealed that the WT and TC PEC/Cowden have 100% nucleotide sequence identities in the 5' terminus, 2C helicase, ORF2, and the 3' nontranslated region. TC PEC/Cowden has one silent mutation in its protease, two amino acid changes and a silent mutation in its RNA polymerase, and five nucleotide substitutions in its capsid that result in one distant and three clustered amino acid changes and a silent mutation. These substitutions may be associated with adaptation of TC PEC/Cowden to cell culture. The cultivable PEC should be a useful model for studies of the pathogenesis, replication, and possible rescue of uncultivable human enteric caliciviruses. (+info)
A review of virus infections of cataceans and the potential impact of morbilliviruses, poxviruses and papillomaviruses on host population dynamics.
Viruses belonging to 9 families have been detected in cetaceans. We critically review the clinical features, pathology and epidemiology of the diseases they cause. Cetacean morbillivirus (family Paramyxoviridae) induces a serious disease with a high mortality rate and persists in several populations. It may have long-term effects on the dynamics of cetacean populations either as enzootic infection or recurrent epizootics. The latter presumably have the more profound impact due to removal of sexually mature individuals. Members of the family Poxviridae infect several species of odontocetes, resulting in ring and tattoo skin lesions. Although poxviruses apparently do not induce a high mortality, circumstancial evidence suggests they may be lethal in young animals lacking protective immunity, and thus may negatively affect net recruitment. Papillomaviruses (family Papovaviridae) cause genital warts in at least 3 species of cetaceans. In 10% of male Burmeister's porpoises Phocoena spinipinnis from Peru, lesions were sufficiently severe to at least hamper, if not impede, copulation. Members of the families Herpesviridae, Orthomyxoviridae and Rhabdoviridae were demonstrated in cetaceans suffering serious illnesses, but with the exception of a 'porpoise herpesvirus' their causative role is still tentative. Herpes-like viruses and caliciviruses (Caliciviridae) give rise to cutaneous diseases in Monodontidae and Delphinidae. Antibodies to several serotypes of caliciviruses were found in odontocetes and mysticetes. An unrecognized Hepadnaviridae was detected by serology in a captive Pacific white-sided dolphin Lagenorhynchus obliquidens with chronic persistent hepatitis. Adenoviruses (Adenoviridae) were isolated from the intestinal tracts of mysticeti and a beluga Delphinapterus leucas but were not associated with any pathologies. We discuss the potential impact of Paramyxoviridae, Poxviridae and Papovaviridae on the dynamics of several odontocete populations. (+info)
Development of methods to detect "Norwalk-like viruses" (NLVs) and hepatitis A virus in delicatessen foods: application to a food-borne NLV outbreak.
"Norwalk-like viruses" (NLVs) and hepatitis A virus (HAV) are the most common causes of virus-mediated food-borne illness. Epidemiological investigations of outbreaks associated with these viruses have been hindered by the lack of available methods for the detection of NLVs and HAV in foodstuffs. Although reverse transcription (RT)-PCR methods have been useful in detecting NLVs and HAV in bivalve mollusks implicated in outbreaks, to date such methods have not been available for other foods. To address this need, we developed a method to detect NLVs and HAV recovered from food samples. The method involves washing of food samples with a guanidinium-phenol-based reagent, extraction with chloroform, and precipitation in isopropanol. Recovered viral RNA is amplified with HAV- or NLV-specific primers in RT-PCRs, using a viral RNA internal standard control to identify potential sample inhibition. By this method, 10 to 100 PCR units (estimated to be equivalent to 10(2) to 10(3) viral genome copies) of HAV and Norwalk virus seeded onto ham, turkey, and roast beef were detected. The method was applied to food samples implicated in an NLV-associated outbreak at a university cafeteria. Sliced deli ham was positive for a genogroup II NLV as determined by using both polymerase- and capsid-specific primers and probes. Sequence analysis of the PCR-amplified capsid region of the genome indicated that the sequence was identical to the sequence from virus detected in the stools of ill students. The developed method is rapid, simple, and efficient. (+info)
Expression and processing of the canine calicivirus capsid precursor.
The ORF2 product of canine calicivirus (CaCV) was identified and its processing in mammalian cells was analysed. Immunoblot analysis revealed the presence of the 75 kDa capsid precursor in addition to a 57 kDa capsid protein and a 22 kDa N-terminal polypeptide in CaCV-infected cells treated at an elevated temperature. When the CaCV ORF2 was expressed in a transient mammalian expression system, only the 75 kDa precursor was detected in immunoblot analysis, suggesting that no post-translational processing occurred in this system. However, the precursor was processed to a 57 kDa protein and a 22 kDa polypeptide by the proteinase of feline calicivirus (FCV) when this was co-expressed with ORF2. Processing was blocked by site-directed mutagenesis of the putative cleavage site in the capsid precursor. The results indicate that the proteinase of FCV can cleave the capsid precursor of CaCV to produce the mature capsid protein and that CaCV may have a similar proteinase. (+info)