Takaaki Sugimura

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.

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.

Evaluation of glycoprotein gII ISCOMs subunit vaccine for pseudorabies in pig

One of the pseudorabies virus glycoproteins, gII, was integrated into Quil A matrix and formed an antigenic presentation structure known as immunostimulating complexes (ISCOMs). The gII ISCOMs protected mice from a lethal infection of pseudorabies virus (PrV). Subsequently, the efficacy of gII ISCOMs as a vaccine was evaluated in pigs, a natural host species of PrV. Immunization of pigs with gII ISCOMs protected them from PrV-induced acute symptoms and death. The serum obtained from immunized pigs was clearly discriminated from that of infected pigs by immunoprecipitation. The data indicated that the ISCOM technique was useful in PrV subunit vaccine preparation and demonstrated the importance of glycoprotein gII as a component of PrV vaccine.

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.