Canine Calicivirus Research Articles

Molecular survey of parvovirus, astrovirus, coronavirus, and calicivirus in symptomatic dogs.

Gastrointestinal disorders caused by enteric viruses are frequently reported in dogs worldwide, with significant mortality rates in unvaccinated individuals. This study reports the identification and molecular characterization of Canine parvovirus (CPV-2), Canine coronavirus (CcoV), Canine astrovirus (AstV), and Canine calicivirus (CcaV) in a panel of dogs showing severe enteric clinical signs sampled in a typical Mediterranean environment (Sardinia, Italy). At least one of these viral species was detected in 92.3% samples. CPV-2 was the most frequently detected virus (87.2%), followed by AsTv (20.5%), CCoV-IIa (18%), and CCoV-I (10.3%). CCoV-IIb and CaCV were not detected in any sample. Single infection was detected in 24 samples (66.7%), mainly related to CPV-2 (91.7%). Coinfections were present in 33.3% samples with constant detection of CPV-2. Canine coronavirus was present only in coinfected animals. The VP2 sequence analysis of CPV-2 positive samples confirmed the presence of all variants, with CPV-2b most frequently detected. Phylogeny based on the CcoV-IIa spike protein (S) gene allowed to identify 2 different clades among Sardinian isolates but failed to distinguish enteric from pantropic viruses. Study on presence and prevalence of enteroviruses in dogs increase our knowledge about the circulation of these pathogens in the Mediterranean area and highlight the need for dedicated routine vaccine prophylaxis. Molecular analyses of enteric viruses are fundamental to avoid failure of vaccines caused by frequent mutations observed in these enteroviruses.

Canine caliciviruses of four serotypes from military and research dogs recovered in 1963\u22121978 belong to two phylogenetic clades in the Vesivirus genus

BackgroundVesiviruses (family Caliciviridae) had been shown capable of invading a variety of host species, raising concern of their zoonotic potential. Since the 1980’s, several canine caliciviruses (CaCV) isolates have been reported and are phylogenetically related to the vesiviruses with features distinct from both Vesicular exanthema of swine virus (VESV) and Feline calicivirus (FCV) species in phylogeny, serology and cell culture specificities. Etiological studies of canine diseases in dogs used for military services and laboratory studies were conducted in 1963–1978 at the Walter Reed Army Institute of Research. Multiple known and unknown viral pathogens including caliciviruses were recovered.MethodsFour unidentified isolates were recovered in Walter Reed Canine Cells (WRCC) from respiratory, fecal and penile specimens. Physicochemical tests, electron microscopy, viral cultivation in human and animal cells, antibody neutralization assays, and recently the genome sequencing were used to characterize the isolates. Sera from these dogs and their cohorts were tested with the isolates to determine origin and prevalence of the infections.ResultsThe viral isolates were small non-enveloped spherical RNA virions, 27 to 42 nm in diameter with cup-like structures, indicating they are caliciviruses. They propagated in WRCC and MDCK cells, not in either other canine cells or human and other animal cells. Each isolate is antigenically distinct and react with dog sera in respective cohorts. The genomes have nucleotide identities ranging from 70.3% to 90.7% and encode the non-structural polyprotein (1810 amino acids), major capsid protein (691 amino acids) and minor structural protein (134 amino acids). They belong to two different phylogenetic clades in Vesivirus genus with close relation with canine calicivirus (CaCV).ConclusionsThese CaCV isolates have restricted cell tropism, antigenic diversity and genetic variation. Further investigation will shed light on antigenic relation to other vesiviruses, and its pathogenicity for dogs and potential infectivity to other animals. Together with the previously reported CaCV strains provides significant evidence to support the formation of a new CaCV species in the Vesivirus genus.

Potential internalisation of caliciviruses in lettuce

Fresh produce such as lettuce ( Lactuca sativa) has often been linked to epidemic viral gastroenteritis. In these cases, it is unknown whether the viral contamination has occurred during the growing or the processing of the implicated product. In this study lettuce was grown in the presence of enteric viruses, and the uptake of viruses via the roots into the edible parts (leaves and stem) of the lettuce plants was investigated, for plants with both intact and damaged roots. The roots of lettuce, growing either in hydroponic culture or in soil, were exposed to canine calicivirus (CaCV) and a human genogroup 2 norovirus (HuNoV) by these being added into the water or soil in which the lettuce was growing. Leaves from lettuce plants and seedlings were examined for viruses by real-time RT-PCR. When the lettuce plants were exposed to very high concentrations of CaCV, the virus was detected in lettuce leaves, indicating contamination via the roots, but the frequency of positive results was low. Internalisation occurred in both seedlings and grown plants, in both hydroponic and soil cultures, and occurred whether the roots were intact or damaged. However, internalisation of HuNoV was not detected in any of the experimental set ups, although the concentrations to which the plants were exposed were relatively high. Based on these results, viral contamination of lettuce plants via roots cannot be excluded, but is apparently not an important transmission route for viruses.

Survival of Mycobacterium avium , Legionella pneumophila , Escherichia coli , and Caliciviruses in Drinking Water-Associated Biofilms Grown under High-Shear Turbulent Flow

Most of the bacteria in drinking water distribution systems are associated with biofilms. In biofilms, their nutrient supply is better than in water, and biofilms can provide shelter against disinfection. We used a Propella biofilm reactor for studying the survival of Mycobacterium avium, Legionella pneumophila, Escherichia coli, and canine calicivirus (CaCV) (as a surrogate for human norovirus) in drinking water biofilms grown under high-shear turbulent-flow conditions. The numbers of M. avium and L. pneumophila were analyzed with both culture methods and with peptide nucleic acid fluorescence in situ hybridization (FISH) methods. Even though the numbers of pathogens in biofilms decreased during the experiments, M. avium and L. pneumophila survived in biofilms for more than 2 to 4 weeks in culturable forms. CaCV was detectable with a reverse transcription-PCR method in biofilms for more than 3 weeks. E. coli was detectable by culture for only 4 days in biofilms and 8 days in water, suggesting that it is a poor indicator of the presence of certain waterborne pathogens. With L. pneumophila and M. avium, culture methods underestimated the numbers of bacteria present compared to the FISH results. This study clearly proved that pathogenic bacteria entering water distribution systems can survive in biofilms for at least several weeks, even under conditions of high-shear turbulent flow, and may be a risk to water consumers. Also, considering the low number of virus particles needed to result in an infection, their extended survival in biofilms must be taken into account as a risk for the consumer.

Detection of Noroviruses in Foods: A Study on Virus Extraction Procedures in Foods Implicated in Outbreaks of Human Gastroenteritis

Disease outbreaks in which foods are epidemiologically implicated as the common source are frequently reported. Noroviruses and enteric hepatitis A viruses are among the most prevalent causative agents of foodborne diseases. However, the detection of these viruses in foods other than shellfish is often time-consuming and unsuccessful. In this study, three virus concentration methods were compared: polyethylene glycol (PEG) plus NaCl, ultracentrifugation, and ultrafiltration. Two RNA extraction methods, TRIzol and RNeasy Mini Kit (Qiagen), were compared for detection of viruses in whipped cream and lettuce (as representatives of the dairy and vegetable-fruit food groups, respectively). A seeding experiment with canine calicivirus was conducted to determine the efficiency of each virus extraction procedure. The PEG-NaCl-TRIzol method was most efficient for the detection of viruses in whipped cream and the ultracentrifugation–RNeasy–Mini Kit procedure was best for detection on lettuce. Based on the seeding experiments, food items implicated in norovirus-associated gastroenteritis outbreaks were subjected to the optimal procedure for a specific composition and matrix. No noroviruses were detected in the implicated food items, possibly because the concentration of virus on the food item was too low or because of the presence of inhibitory factors. For each food group, a specific procedure is optimal. Inhibitory factors should be controlled in these procedures because they influence virus detection in food.

Real-time reverse transcription polymerase chain reaction assay to detect a broad range of feline calicivirus isolates

This report describes a real-time reverse transcription polymerase chain reaction (RT-PCR) assay with SYBR ® Green targeting the VP2 (ORF 3) of feline caliciviruses. All of the 44 feline calicivirus isolates tested were detected, whereas neither feline herpesvirus, feline panleukopenia virus, feline immunodeficiency virus and feline leukemia virus nor other calicivirus like rabbit hemorrhagic disease virus and a canine calicivirus isolate showed specific amplification products. The sensitivity of the SYBR ® Green reaction was shown to be equivalent to 5 × 10 1 to 5 × 10 2 copies/reaction and the overall sensitivity equivalent to a feline calicivirus titer of 10 0.6 TCID 50/100 μl in Crandell Reese Feline Kidney (CRFK) cells.

Calicivirus inactivation by nonionizing (253.7-nanometer-wavelength [UV]) and ionizing (gamma) radiation.

Noroviruses (previously Norwalk-like viruses) are the most common viral agents associated with food- and waterborne outbreaks of gastroenteritis. In the absence of culture methods for noroviruses, animal caliciviruses were used as model viruses to study inactivation by nonionizing (253.7-nm-wavelength [UV]) and ionizing (gamma) radiation. Here, we studied the respiratory feline calicivirus (FeCV) and the presumed enteric canine calicivirus (CaCV) and compared them with the well-studied bacteriophage MS2. When UV irradiation was used, a 3-log(10) reduction was observed at a fluence of 120 J/m(2) in the FeCV suspension and at a fluence of 200 J/m(2) for CaCV; for the more resistant phage MS2 there was a 3-log(10) reduction at a fluence of 650 J/m(2). Few or no differences were observed between levels of UV inactivation in high- and low-protein-content virus stocks. In contrast, ionizing radiation could readily inactivate MS2 in water, and there was a 3-log(10) reduction at a dose of 100 Gy, although this did not occur when the phage was diluted in high-protein-content stocks of CaCV or FeCV. The low-protein-content stocks showed 3-log(10) reductions at a dose of 500 Gy for FeCV and at a dose of 300 for CaCV. The inactivation rates for both caliciviruses with ionizing and nonionizing radiation were comparable but different from the inactivation rates for MS2. Although most FeCV and CaCV characteristics, such as overall particle and genome size and structure, are similar, the capsid sequences differ significantly, making it difficult to predict human norovirus inactivation. Adequate management of UV and gamma radiation processes for virus inactivation should limit public health risks.

Inactivation of caliciviruses.

The viruses most commonly associated with food- and waterborne outbreaks of gastroenteritis are the noroviruses. The lack of a culture method for noroviruses warrants the use of cultivable model viruses to gain more insight on their transmission routes and inactivation methods. We studied the inactivation of the reported enteric canine calicivirus no. 48 (CaCV) and the respiratory feline calicivirus F9 (FeCV) and correlated inactivation to reduction in PCR units of FeCV, CaCV, and a norovirus. Inactivation of suspended viruses was temperature and time dependent in the range from 0 to 100 degrees C. UV-B radiation from 0 to 150 mJ/cm(2) caused dose-dependent inactivation, with a 3 D (D = 1 log(10)) reduction in infectivity at 34 mJ/cm(2) for both viruses. Inactivation by 70% ethanol was inefficient, with only 3 D reduction after 30 min. Sodium hypochlorite solutions were only effective at >300 ppm. FeCV showed a higher stability at pH <3 and pH >7 than CaCV. For all treatments, detection of viral RNA underestimated the reduction in viral infectivity. Norovirus was never more sensitive than the animal caliciviruses and profoundly more resistant to low and high pH. Overall, both animal viruses showed similar inactivation profiles when exposed to heat or UV-B radiation or when incubated in ethanol or hypochlorite. The low stability of CaCV at low pH suggests that this is not a typical enteric (calici-) virus. The incomplete inactivation by ethanol and the high hypochlorite concentration needed for sufficient virus inactivation point to a concern for decontamination of fomites and surfaces contaminated with noroviruses and virus-safe water.

Evaluation of a p30 Gene‐Based Real‐Time Reverse Transcriptase Polymerase Chain Reaction Assay for Detection of Feline Caliciviruses

This report describes a feline calicivirus (FCV) p30 gene‐based real‐time SYBR Green I reverse transcriptase polymerase chain reaction (RT‐PCR) assay that is capable of detecting low virus concentrations and a broad range of FCV isolates. The assay consisted of a 1‐step RT‐PCR reaction with primers delineating a 126‐base‐pair (bp) region of the FCV p30 gene. Sensitivity of the RT‐PCR assay was determined to be equivalent to a FCV titer of 1.2 × 101 to 1.2 × 102 TCID50/mL. The assay was linear over a wide range of template concentrations and had a reaction efficiency of 95%. Specific FCV amplification products were detected from 51 wild‐type FCV isolates, whereas specific products were not detected from a canine calicivirus, a rabbit calicivirus, and a bovine calicivirus. The primers used in this study amplified a large number of North American FCV isolates and further confirm the diagnostic utility of p30 gene‐based real‐time RT‐PCR for detection of FCV.

Identification of a calicivirus isolate of unknown origin.

Chinese hamster ovary (CHO) cells manifesting striking cytopathogenic changes in culture were investigated to determine the causative agent. Electron microscopic analyses revealed viral particles of about 40 nm in diameter, displaying typical calicivirus morphology. To date, this virus, designated isolate 2117, exclusively replicates in CHO cells, achieving only moderate titres. After cloning, the coding region of 7928 nucleotides, the 3' non-coding region and the poly(A) tail were sequenced. The genome consists of three open reading frames (ORFs), with the first and second ORF having the same reading frame. The overall genomic organization as well as the nucleotide sequence of isolate 2117 is most similar to that of a recently described canine calicivirus, but also shows significant similarity to the sequences of mink calicivirus and other caliciviruses within the genus Vesivirus: In Western blots, using antibodies against the viral protease, a stable, unprocessed 3CD protein of 68 kDa was identified in homogenates of 2117-infected CHO cells. Furthermore, antibodies raised against ORF 3 reacted with the respective protein in 2117-virions, demonstrating that this predicted 9 kDa protein is a minor structural component of the virion. In addition, an RT-PCR assay was established to detect 2117 viral RNA in biological products such as foetal bovine serum, which will aid the discovery of the origin and host of the virus.

Molecular and seroepidemiological evidence of canine calicivirus infections in Japan.

Calicivirus infection of dogs was epidemiologically investigated by using canine calicivirus (CaCV) strain 48 as a reference. Similar RNA polymerase gene sequences and neutralizing antibodies against CaCV were detected in 1.7% of clinical specimens and 57% of serum samples, respectively, suggesting a high prevalence of CaCV in dog populations.

Early interaction of canine calicivirus with cells is the major determinant for its cell tropism in vitro

Canine calicivirus (CaCV) No. 48 strain isolated from a dog with fatal diarrhea is known to be able to replicate in MDCK and primary dog kidney cells. In this study, two new canine cell lines, MCM-B2 and MCA-B1, were determined to be permissive for CaCV No. 48, whereas other cell lines, including one canine cell line, A-72, were non-permissive. Flow cytometric analysis indicated that CaCV No. 48 binds efficiently to the permissive cells and to some degree also to Vero cells that are non-permissive for the virus, but does not bind to the other non-permissive cells tested. Both the permissive and non-permissive cells could be transfected with genomic RNA from CaCV No. 48, resulting in the appearance of CPE, production of capsid antigen and release of infectious progeny. These results suggested that the early interaction of the virus with cells, probably by binding to a virus receptor on the cell membrane, is the major determinant of CaCV No. 48 cell tropism in vitro.

Complete nucleotide sequence, genome organization and phylogenic analysis of the canine calicivirus.

The complete genomic sequence of canine calicivirus (CaCV) isolated from feces of a dog with diarrhea was determined. The CaCV genome, a positive-sense single-stranded RNA, contained 8513 nucleotides excluding the poly(A) tail and was longer than that of any other calicivirus strain with a completely known sequence. There were three open reading frames (ORF1, nt 12-5801; ORF2, nt 5805-7880; and ORF3, nt 7877-8278). ORF1 encoded a polyprotein (calculated Mr of 214,802) which had the conserved motifs of non-structural proteins of other caliciviruses and picornaviruses. Regions containing characteristic motifs in the non-structural polyprotein of CaCV showed highest similarity with those of the species Feline calicivirus and Vesicular exanthema of swine virus in the genus Vesivirus. Phylogenic analysis indicated that CaCV formed a distinct branch within the genus. Our results strongly suggested that CaCV is a new species in the genus Vesivirus.

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.

Genetic analysis of a canine calicivirus: evidence for a new clade of animal caliciviruses

This paper describes the relationship of a canine calicivirus, named No.48, to other human and animal caliciviruses, based on phylogeny of the 3′ half of its genome. It was found that No.48 constitutes a unique lineage, most closely related but distinct from feline and San Miguel sea lion caliciviruses.

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.

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.

Identification of canine calicivirus capsid protein and its immunoreactivity in western blotting.

A canine calicivirus (CaCV) isolated in Japan, designated as CaCV No. 48 strain, was propagated in MDCK cells and purified by CsCl equilibrium gradient centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified samples revealed the presence of only one major species of viral protein of about 60 kilodaltons after Coomassie staining. The same band, presumably that of the capsid protein, was detected by western blotting using a mouse hyperimmune serum. This capsid protein was synthesized in MDCK cells as early as 2 hr post-inoculation. Experimental infection of dogs resulted in the production of anti-CaCV antibodies which were detected by microneutralization test and western blotting. Likewise, serosurvey revealed not only the presence of neutralizing antibodies but also reactivity of the field sera against the capsid protein of the purified virus. These results indicate that the capsid protein of CaCV No. 48 strain is immunogenic and could be detected by antibodies in western blotting.

Isolation of a calicivirus antigenically related to feline caliciviruses from feces of a dog with diarrhea.

A CPE-producing agent was recovered in feline cell cultures from feces of a male dog suffering from intermittent watery diarrhea. Antigenic analysis of this isolate, Sapporo/283, was performed using the plaque reduction neutralization and complement fixation assays and it was neutralized by antisera against feline calicivirus (FCV) but not against canine calicivirus (CaCV). Likewise, it showed common CF antigenicity with the other FCV strains included in the experiments. These findings revealed that the isolate was more closely related antigenically to FCV than CaCV, indicating the possibility of interspecies transmission. It was also suggested that the isolate was a respiratory type calicivirus. Epizootiological results suggested, however, that FCV seldom infects dogs under natural condition.

Characterization of a new calicivirus isolated from feces of a dog.

Canine calicivirus (CaCV), isolated from feces of a dog with diarrhea, was readily propagated in cultures of canine cells and in a dolphin cell line. Serologic evidence indicated many dogs in at least one geographic area had been infected with CaCV, but its role as an etiologic agent of disease was not established. In cell culture most CaCV virions were strongly cell-associated making purification difficult. CaCV was established as a member of the Caliciviridae by morphology and physicochemical properties of virions (density, sedimentation rate, single major polypeptide, RNA genome size), although some of the properties differed slightly from those of previously described caliciviruses; evidence was also obtained for caliciviral RNA species in infected cells. Based on tests with antisera to numerous caliciviruses and presumed caliciviruses, CaCV appeared to be not closely related to any previously described virus except the stunting syndrome agent of chickens.