Naoaki Yokoyama

In vitro recombination of feline herpesvirus type 1

SummaryWe investigated the possibility of in vitro recombination of three different trains of feline herpesvirus type 1 (FHV-1), a virulent strain (C7805), a vaccine strain (F2), and a candidate vaccine strain with a deletion in the thymidine kinase gene (C7301dlTK). The results showed that recombination within different genotypes of FHV-1 strains could occur at a relatively high frequency ranging from 10.1% to 21.5% in vitro. These results indicate the generation of multiple mutant viruses by in vitro recombination experiments. The possibility for generating a virulent virus or novel type of FHV-1 by recombination in vivo is discussed.

Pathogenicity and vaccine efficacy of a thymidine kinase-deficient mutant of feline herpesvirus type 1 in cats

SummaryWe constructed a recombinant feline herpesvirus type 1 (FHV-1) which was deleted in a defined region (450 bp) within the thymidine kinase (TK) gene (C7301dlTK) [Yokoyama et al. (1995) J Vet Med Sci 57: 709–714]. In this report, we carried out two experiments to assess the pathogenicity and vaccine efficacy of the recombinant C7301dlTK in cats. The first experiment showed that, following multiple inoculation of the recombinant C7301dlTK by intraocular, intranasal and oral routes, the virus was sufficiently attenuated in cats, although a high titer of the virus was recovered from target organs (eye, nose, and mouth). In the second experiment, two intramuscular vaccinations with the recombinant C7301dlTK protected cats to a significant degree against subsequent challenge with the parent FHV-1 strain C7301 at 4 weeks after the last vaccination. These results demonstrate that the recombinant C7301dlTK is effective as a live attenuated vaccine with a clear genetic marker.

Vaccine efficacy of recombinant feline herpesvirus type 1 expressing immunogenic proteins of feline calicivirus in cats

SummaryWe previously constructed a recombinant feline herpesvirus type 1 (FHV1), C7301dlTK-Cap, which contains an entire open reading frame encoding the capsid protein of feline calicivirus (FCV) F4 strain in the deleted locus of the thymidine kinase (TK) deficient mutant (C7301dlTK) of FHV 1. In this report, we carried out in vivo experiments to assess the vaccine efficacy of the recombinant C7301dlTK-Cap against FCV and FHV 1 infections in cats. As a result, two vaccinations with the C7301dlTK-Cap by intraocular, intranasal and oral routes protected cats to a significant degree against subsequent virulent challenges with both parent FCV F4 and FHV 1 C7301 strains. The results are applicable for the further development of a new genetically engineered polyvalent vaccine for cats.

Expression and identification of the feline herpesvirus type 1 glycoprotein B (gp143/108)

The gene for feline herpesvirus type 1 (FHV-1) glycoprotein B (gB) has been cloned into an expression vector, pRVSVneo, containing the long terminal repeat of Rous sarcoma virus and polyadenylation signal of SV40. This expression vector containing FHV-1 gB gene, pRVSVgBneo, was transfected into Crandell feline kidney (CRFK) cells which are susceptible to FHV-1 infection. By indirect immunofluorescence analysis, the expressed gB was recognized with a panel of monoclonal antibodies (MAbs) against FHV-1 gp143/108. Immunoprecipitation analysis using a MAb 34H12 showed that molecular weights of the gB were 143 and 108 kDa under non-denaturing conditions that 108, 70, 64, and 58 kDa under denaturing conditions. The molecular weights were similar to those of the gB expressed in FHV-1-infected CRFK cells. In addition, when plasmid DNAs were injected into mice to obtain gB-monospecific serum, the pooled serum from mice inoculated with pRVSVgBneo, but not with pRVSVgDneo or pRVSVneo, recognized the FHV-1 gB polypeptides.

Expression and properties of feline herpesvirus type 1 gD (hemagglutinin) by a recombinant baculovirus

We constructed a recombinant baculovirus expressing feline herpesvirus type I (FHV-1) gD in insect cells (Sf9 cells). The expressed product was identified as FHV-1 gD by a panel of monoclonal antibodies specific for the FHV-1 gD, and had an apparent molecular mass of approximately 49 kDa, which was less than that of the authentic FHV-1 gD. When the FHV-1 gD protein were expressed in Sf9 cells and CRFK cells in the presence of tunicamycin, the FHV-1 gD exhibited a molecular mass of 41 kDa. It was shown that the gD protein was transported to the surface of recombinant virus-infected Sf9 cells when examined by membrane-immunofluorescence analysis, and that the gD expressed on the surface of Sf9 cells adsorbed feline erythrocytes. Mice inoculated with a lysate of Sf9 cells expressing FHV-1 gD induced antibodies with virus-neutralizing and hemagglutination-inhibition activities. Therefore, the expressed gD appears to be biologically authentic. These data suggested that recombinant FHV-1 gD produced in Sf9 cells may be a useful immunogen as a feline vaccine.

Construction of a recombinant feline herpesvirus type 1 expressing Gag precursor protein of feline immunodeficiency virus

SummaryWe constructed a deletion mutant of feline herpesvirus type 1 (FHV-1) and a recombinant FHV-1. The deletion mutant is the virus with a region (367 bp) deleted from the start codon of thymidine kinase (TK) gene to the SmaI site within the TK gene, and the other is a recombinant FHV-1 expressing Gag protein of feline immunodeficiency virus (FIV), in which a cDNA encoding the Gag protein of FIV was inserted at the TK deletion site of the former deletion mutant. These viruses were designated as C7301ddlTK and C7301ddlTK-gag, respectively. Growth kinetics of these viruses in Crandell feline kidney cells was similar to that of the parent C7301 strain. By immunoblot analysis, C7301ddlTK- gag was confirmed to express the FIV Gag precursor protein in the cells.

Heparin-binding activity of feline herpesvirus type 1 glycoproteins.

Feline herpesvirus type 1 (FHV-1) possesses a very narrow host range, but the mechanism of its infection has not yet been analyzed. Heparan sulfate on the cell surface serves as a receptor for several herpesviruses. In this study, we determined that infection of FHV-1 is inhibited by addition of soluble heparin in cells cultures. Using heparin-affinity column, it was shown that FHV-1 gC is a major heparin-binding protein, and FHV-1 gB weakly binds to heparin, but FHV-1 gD does not. Furthermore, the FHV-1 gC expressed in insect cells can also bind to heparin despite of being immature glycosylation. Our results suggested that FHV-1 gC can bind to heparin as observed in other herpesviruses and that glycosylation of the gC does not affect its heparin-binding activity. In addition, mice immunized with the gC expressed in insect cells produced complement-dependent virus-neutralizing antibody.

Identification and characterization of the feline herpesvirus type 1 glycoprotein C gene.

The feline herpesvirus type 1 (FHV-1) gene encoding glycoprotein C (gC) has been sequenced and identified based on its genomic location and comparative analysis to other alphaherpesvirus gCs, and the expressed gC protein was also identified by using specific monoclonal antibodies. The FHV-1 gC gene was located within a 7.0 kbp EcoRI fragment, and was 1602 bp in length. The amino acid sequence deduced from the nucleotide sequence was predicted to encode a membrane glycoprotein containing a characteristic N-terminal hydrophobic signal sequence, nine potentialN -linked glycosylation sites, and C-terminal transmembrane and cytoplasmic domains. The FHV-1 gC was expressed in COS-7 cells. When flowcytometric analysis was carried out, the gC expressed in COS-7 cells reacted with a panel of monoclonal antibodies against gp113. By immunoprecipitation analysis, the gC expressed in COS-7 cells possessed molecular masses of 125–;150 kilodalto n, and was similar in size to that in FHV-1-infected CRFK cells.

Protection studies against Marek's disease using baculovirus‐expressed glycoproteins B and C of Marek's disease virus type 1

Glycoproteins B (MDV1-gB) and C (MDV1-gC) of Mareks disease virus type 1 (MDV 1) expressed by baculovirus recombinants [rAcNPV(s)] were investigated for their ability to confer protective immunity in chickens against virulent MDV1 challenge. Immunization of chickens with rAcNPV-expressed MDV1-gB induced antibody against MDV1 and resulted in strong protection against the lethal challenge with MDV1. In contrast, immunization of chickens with rAcNPV-expressed MDV1-gC induced antibody against MDV1, but failed to protect against challenge with the virulent MDV1. Co-immunization with both rAcNPV-expressed MDV1-gB and MDV1-gC seemed to show no synergistic effect.

Construction of Canine Herpesvirus Vector Expressing Foreign Genes Using a lacZ-TK Gene Cassette as a Double Selectional Marker

An improved method for constructing canine herpesvirus (CHV) recombinants expressing foreign genes by using the lacZ-TK gene cassette as a double selectional marker was developed. A recombinant CHV carrying the lacZ-TK gene at a targeted gene locus was constructed and used as a parental virus for generating new recombinants. The parental virus formed blue plaques and was sensitive to TK-specific drugs, while newly generated recombinants, in which the lacZ-TK gene was replaced with the desired foreign gene, become both resistant to the TK-specific drugs and formed white plaques. Recombinants were isolated by using the combination of drug selection and color selection. This improved method allows construction of recombinant CHV with great ease, because the drug selection can enrich the frequency of recombinant CHV from 0.01–0.1% to 10–80%. This method was employed to construct a recombinant CHV that expressed rabies virus (RV) glycoprotein (G protein).