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Feline calicivirus (FCV) causes upper respiratory tract diseases in cats and has highly variable antigenicity for neutralization of each strain. Neutralizing epitopes of FCV are currently found in the hypervariable region (HVR) in the P2 domain of the major capsid protein VP1. Due to its unique ability to neutralize various FCV strains, 1D7 is a monoclonal antibody that may recognize a novel neutralizing epitope. While other neutralizing epitopes were characterized by producing neutralization-resistant variants, only 1D7-resistant variants could not be obtained, and its epitope has not been identified in the previous studies. In this study, we successfully generated these variants by multiple passaging of the FCV F4 strain in the presence of 1D7 and discovered that several amino acid substitutions (K638N, R662G, and T666I in the P1 domain of VP1) are involved in the decreased binding of 1D7. These substitution sites are also highly conserved among FCV strains compared with the substitution sites of other neutralization-resistant variants found in the HVR. Our results indicate that amino acid substitutions in the P1 domain, which are not responsible for direct interaction with the FCV receptor, are associated with neutralization escape. Since FCV can be conveniently cultured in vitro and the receptor required for infection is known, a detailed analysis of the 1D7 epitope could shed more light on the neutralization mechanism of the epitopes of viruses belonging to the Caliciviridae., (Copyright © 2022 Elsevier B.V. All rights reserved.)

A novel calicivirus, St-Valerien-like virus (SVV), has been identified in asymptomatic swine in Canada, Italy and the U.S.A. In this study, we characterized a new SVV strain (NUP-24/JP) detected in fecal samples of swine in Japan. The NUP-24/JP genome had 6,409 nucleotides and 2 open reading frames (ORF1 and ORF2). ORF1 and ORF2 consist of 5,940 and 453 nucleotides, respectively. Phylogenetic analysis revealed that NUP-24/JP was closely related to other SVV strains, particularly to U.S.A. strain NC-WGS93C/US. This finding suggests that SVV is prevalent in swine worldwide. Using a baculovirus expression system, we successfully produced virus-like particles, which would be useful for seroepidemiological studies of SVV.

Enteric caliciviruses, noroviruses, and sapoviruses are emerging pathogens responsible for diarrhea or gastroenteritis in their respective hosts. In this study, swine enteric caliciviruses were detected in ten samples of intestinal contents from 24 piglets in Japan by reverse transcription-polymerase chain reaction using a broadly reactive primer pair (P290/289) that targeted the highly conserved RNA polymerase regions of the enteric caliciviruses. From the positive samples, the entire viral genome of strain K7/JP and 3'-end parts of the genomes of strains K5/JP and K10/JP were cloned and sequenced. K7/JP had an RNA genome of 7144 bases, excluding its 3' poly (A) tail. The K7/JP genome possessed two open reading frames and characteristics common to sapoviruses. In phylogenetic analysis using amino acid sequences of VP1, K5/JP was demonstrated to be close to the noroviruses previously detected in pigs, and K7/JP and K10/JP were considered to be classified as a new genogroup of sapoviruses.

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.

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.

Two neutralizing monoclonal antibodies (MAbs) against canine calicivirus (CaCV), which has a distinct antigenicity from feline calicivirus (FCV), were obtained. Both MAbs recognized conformational epitopes on the capsid protein of CaCV and were used to identify these epitopes. Neutralization-resistant variants of CaCV were selected in the presence of individual MAbs in a cell culture. Cross-neutralization tests using the variants indicated that the MAbs recognized functionally independent epitopes on the capsid protein. Recombinantly expressed ORF2 products (capsid precursors) of the variants showed no reactivity to the MAbs used for the selection, suggesting that the resistance was induced by a failing in binding of the MAbs to the variant capsid proteins. Several nucleotide changes resulting in amino acid substitutions in the capsid protein were found by sequence analysis. Reactivities of the MAbs to the revertant ORF2 products produced from each variant ORF2 by site-directed mutagenesis identified a single amino acid substitution in each variant capsid protein responsible for the failure of MAb binding. The amino acid residues related to forming the conformational neutralizing epitopes were located in regions equivalent to the 5' and 3' hypervariable regions of the FCV capsid protein, where antigenic sites were demonstrated in previous studies. The recombinant ORF2 products expressed in bacteria failed to induce neutralizing antibody, suggesting that neutralizing antibodies were only generated when properly folded capsid protein was used as an antigen. In CaCV, the conformational epitopes may play a more important role in neutralization than do linear epitopes.

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.

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.

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.

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.

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.

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.

The nucleotide sequence of the 3'-end of the Japanese F4 strain of feline calicivirus (FCV) RNA was determined from a cloned cDNA of 3.5 kbp. We found three open reading frames (ORFs). The largest ORF encoded a 668-amino acid protein of 73,588 Da, which was presumably the capsid precursor protein of FCV and had significant amino acid sequence homology with the VP3 of picornaviruses. A small ORF at the extreme 3'-end was compared with that of the F9 strain of FCV, a vaccine strain originally from the U.S. Highly conserved amino acid sequences were shown, suggesting that this ORF might be functional and encode a putative 106-amino acid protein of 12,153 Da. The other ORF in the 5'-flanking region of the cDNA had consensus amino acid sequences conserved among the RNA-dependent RNA polymerases.

A new collection of eighteen neutralizing monoclonal antibodies (N-MoAbs), raised against feline calicivirus (FCV), was used to analyze neutralizing epitopes of the F4 strain of FCV, the prototype strain of FCV in Japan. By cross-neutralization tests with the 20 FCV strains including Japanese. American, Swiss, and New Zealand isolates, the 18 N-MoAbs were categorized into six groups. One N-MoAb (1 D 7) neutralized all the strains tested: eight N-MoAbs neutralized only FCV-F 4; while the others neutralized the FCV strains in various degrees. In order to confirm this grouping, eight N-MoAbs were used to select neutralization-resistant variants of FCV F4. Although no variant against 1 D 7 was obtained, antigenic variants against other N-MoAbs were obtained. Neutralization tests using these variants revealed that there are six neutralizing epitopes on FCV F4 and that several epitopes are functionally related. One of these epitopes was the same epitope as one of the two epitopes identified by another panel of N-MoAbs we produced previously. Therefore, a total of seven neutralizing epitopes on FCV F4 were identified. Immunoblot analysis indicated that four of the seven epitopes existed on the 67 kDa capsid protein of the virus.

In the culture fluid from cells infected with feline calicivirus (FCV) F4 strain, the infectious and smaller non-infectious subunit particles were detected by complement fixation (CF) test after sucrose gradient centrifugation. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses confirmed the existence of the subunit particles of FCV, and showed that the infectious and subunit particles were mainly composed of 65K capsid protein. The subunit particles were further purified by ion-exchange chromatography and sucrose gradient centrifugation. The purified subunit and infectious particles had the same neutralizing epitope on 65K protein detected by immunoblot analysis with a neutralizing monoclonal antibody. Antigenic comparison between the infectious and subunit particles by the CF tests using an antiserum against heterologous strain of FCV F14 indicated that the subunit particles might have more highly conserved antigens of FCV than the infectious particles.

Fourteen neutralizing monoclonal antibodies (N-MoAbs) were prepared against the F4 strain of feline calicivirus (FCV), the prototype strain of FCV in Japan, and examined for their ability to neutralize FCV isolates. Neutralization-resistant variants of the F4 strain were selected under the presence of 4 individual N-MoAbs in cell culture systems and used in cross-neutralization tests and enzyme-linked immunosorbent assay with all of the 14 N-MoAbs. The results revealed the identification of at least two antigenic determinants on FCV F4: one being more broadly conserved among FCV isolates than the other. Usefulness of antigenic variants resistant to N-MoAbs for analysis of neutralization determinants on FCV was also demonstrated.