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A new bat herpesvirus was detected in the spleen of an insectivorous bat (Hipposideros diadema, family Hipposideridae) collected on Panay Island, the Philippines. PCR analyses were performed using COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOPs) targeting the herpesvirus DNA polymerase (DPOL) gene. Although we obtained PCR products with CODEHOPs, direct sequencing using the primers was not possible because of high degree of degeneracy. Direct sequencing technology developed in our rapid determination system of viral RNA sequences (RDV) was applied in this study, and a partial DPOL nucleotide sequence was determined. In addition, a partial gB gene nucleotide sequence was also determined using the same strategy. We connected the partial gB and DPOL sequences with long-distance PCR, and a 3741-bp nucleotide fragment, including the 3' part of the gB gene and the 5' part of the DPOL gene, was finally determined. Phylogenetic analysis showed that the sequence was novel and most similar to those of the subfamily Gammaherpesvirinae.

Prior to pre-exposure treatment of cats with two mouse-cat chimeric antibodies, FJH2 and F1D7, having neutralizing activity to feline herpesvirus-1 (FHV-1) and cat calicivirus (FCV), respectively, these chimeric antibodies were labeled with (125)I and administered to cats to examine their blood kinetics. Concentrations of the both administered chimeric antibodies in the blood reached maximum at the 48th hour post-administration, and the level was 34% for FJH2 and 54% for F1D7. Then the concentration levels declined gently, and decreased afterwards to 8.2% for FJH2 and 25% for F1D7 on the 20th day post-administration. The blood half-lives of FJH2 and F1D7 were 8.3 days and 10.7 days, respectively. In order to examine effectiveness in pre-exposure treatment of cats with these chimeric antibodies, cats were administered on the 15th day prior to the challenge infections with FHV-1 and FCV by subcutaneous route with 0.5 ml/kg of an FJH-F1D7 mixture being adjusted to contain each chimeric antibody of 10 mg/ml. The cats that received the pre-exposure treatment with the cocktail, showed obvious reductions in manifestations of symptoms caused by those viral infections. The protective effectiveness of the pre-exposure treatment against these viral challenge infections was almost equal to that of the treatment given at right after these challenge infections.

Feline herpesvirus type 1 (FHV-1) produces a single 5.4 kb immediate-early (IE) transcript encoding FHV-1 ICP4 which acts as a trans-acting factor [Kawaguchi et al. (1994) Virology 204: 430-435]. Our earlier study has shown that the FHV-1 IE transcript is spliced in the leader region and the FHV-1 IE gene product (ICP4) down-regulates its own promoter through the region which includes its transcription initiation site [Kawaguchi et al. (1996) J Vet Med Sci 58: 715-721]. Here we investigated FHV-1 ICP4 gene expression throughout FHV-1 productive infection and demonstrated that (i) a novel promoter is located downstream of the IE promoter and an early transcript is transcribed from this promoter region, (ii) a negative regulatory element, which is composed of a 20 bp direct repeat unit repeated 20 times, is located between the two promoters and the repeat unit shows high homology to a motif called direct repeat 2 in "a" sequence of herpes simplex virus type 1, (iii) the IE promoter and synthesis of the IE mRNA appear to be turned off after IE phase and the second promoter becomes active during early and late stages, and (iv) a gene product expressed only by the second promoter also possesses regulatory function. These findings indicate that expression of the FHV-1 ICP4 gene is alternatively regulated by the two promoters.

In this study, an entire open reading frame encoding the capsid protein of feline calicivirus (FCV) F4 strain was inserted into the deletion locus (SmaI site) of the thymidine kinase (TK) deficient mutant (C7301dlTK) of feline herpesvirus type 1 (FHV-1) and the resulting recombinant virus was designated as C7301dlTK-Cap. Expression of the FCV antigens by C7301dlTK-Cap was confirmed by indirect immunofluorescence assay and immunoblot analysis. To assess whether the recombinant virus can induce virus neutralizing (VN) antibody against FCV in the natural host, three cats were inoculated intranasally and orally with C7301dlTK-Cap (two cats) or C7301dlTK (one cat). As a result, sera collected from cats inoculated with the C7301dlTK-Cap possessed VN antibody against FCV. This recombinant virus is expected as a new polyvalent recombinant vaccine against FHV-1 and FCV infections.

We 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 FHV1. In this report, we carried out in vivo experiments to assess the vaccine efficacy of the recombinant C7301dlTK-Cap against FCV and FHV1 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 FHV1 C7301 strains. The results are applicable for the further development of a new genetically engineered polyvalent vaccine for cats.

A gene-encoding glycoprotein D (gD) homologue of feline herpesvirus type 1 (FHV-1) was identified and sequenced. It was located within an EcoRI 4.3-kbp fragment in the middle of the unique short region. The primary translation product of the gD homologous gene is predicted to consist of 374 amino acids with a molecular weight (MW) of 43.2 kDa. Comparative analysis among gD counterparts of other herpesviruses revealed that six cysteine residues which correlate with disulfide bond structure were conserved, and the amino acid sequences had homologies of 32.6% with bovine herpesvirus type 1 gIV, 29.1% with pseudorabies virus gp50, 28.3% with equine herpesvirus type 1 gD, and 22.0% with herpes simplex virus type 1 gD. When the gD homologue was expressed in COS cells and examined by indirect immunofluorescence and immunoprecipitation assays, it was recognized only by monoclonal antibodies (MAbs) reactive with gp60 which have virus-neutralizing and hemagglutination (HA)-inhibition (HI) activities. This expressed product had a MW of approximately 60 kDa and was shown to be a hemagglutinin by HA and HI tests with the MAbs. These results suggest that the gD homologue of FHV-1 is a hemagglutinin.

A gene encoding the glycoprotein H (gH) homologue of feline herpesvirus type 1 was identified and sequenced. It was located immediately downstream of the thymidine kinase gene within an EcoRI 6.6 kbp fragment. In addition, a partial UL21 homologous gene was located downstream of the gH homologous gene. The primary translation product of the gH homologous gene is predicted to consist of 821 amino acids with a molecular weight of 92.5 kDa. It possesses several characteristics typical of transmembrane glycoproteins, including a N-terminal hydrophobic signal sequence, C-terminal transmembrane domain, and putative N-linked glycosylation sites. Analysis of this protein revealed amino acid sequence homologies of 33.1% with equine herpesvirus type 1 (EHV-1) gH, 32.6% with EHV-4 gH, 29.1% with varicella-zoster virus gIII, 28.5% with pseudorabies virus gH, and 25.1% with herpes simplex virus type 1 gH. By Northern blot analysis, one of the transcripts specific for the gH homologous gene might be a mRNA of approximately 3.0 kb.

Anti-idiotypic antibodies (anti-Id Abs) were produced in rabbits after inoculation with two mouse monoclonal antibodies (mAbs) directed against canine herpesvirus (CHV) glycoproteins (gps). One of the mAbs, 12H11, was directed against an epitope on gp 145/112 of CHV which induced virus neutralizing (VN) antibodies and against a cross-reacting epitope on the gp 143/108 of feline herpes-virus type 1 (FHV-1). The other mAb, 11F7, was directed against epitopes on CHV gp47 which induce VN and hemagglutination-inhibition (HAI) antibodies. Using VN-inhibition and HAI-inhibition assays with CHV and FHV-1, the anti-Id Abs obviously inhibited the activities of autologous mAbs, suggesting that anti-Id Abs mimic the epitopes of CHV gp 145/112 or FHV-1 gp 143/108 and CHV gp47 by binding the anti-combining site of the mAbs. These anti-Id Abs, when injected into mice, elicited specific CHV-neutralizing and HAI antibody responses, and one of them also elicited a specific FHV-1-neutralizing antibody response. These data supported the idea that immunization with anti-Id Ab can induce specific VN antibody response, as has been theorized by other workers.

The three glycoproteins each of feline herpesvirus type 1 (FHV-1) and canine herpesvirus (CHV) were purified by affinity chromatography using glycoprotein-specific monoclonal antibodies and used individually or in combination in immunizing mice to determine their relative immunogenicity. All the glycoproteins induced detectable virus neutralizing antibodies to the homologous virus but FHV-1 gp143/108 and its cross-reacting counterpart, CHV gp145/112, elicited the highest titers not only to the homologous virus but to the heterologous virus as well. The production of ELISA antibodies after glycoprotein immunization was variable, while hemagglutination-inhibiting antibodies were produced by only 1 out of 10 FHV-1 gp60-inoculated mice. In general, the antibody titers induced by CHV glycoproteins were lower than those by FHV-1 glycoproteins. These results indicate that these glycoproteins may be useful as subunit vaccines against FHV-1 and CHV infections.

Eighteen canine herpesvirus (CHV) isolates from Japan and two reference strains were compared by restriction endonuclease analysis technique using total DNA extracts from cells infected with the viruses. In order to select the suitable restriction endonucleases for differentiation of CHV isolates, ten enzymes were used and three of them, HindIII, XbaI, and PvuII, were found to be useful for strain differentiation. With these enzymes, CHV isolates from unrelated individuals were readily differentiated from each other. In contrast, all the isolates derived from the same litter were not distinguishable on the basis of restriction cleavage patterns. However, slight mobility shifts were observed among the isolates from the same litter or the same individual. The results showed that this method provides a powerful tool for epidemiological surveys of CHV infection.

Monoclonal antibodies (MoAbs) to feline herpesvirus type 1 (FHV-1) were produced to identify the virus-specified immunogenic proteins. When antigens recognized by the MoAbs were investigated by immunoblot and immunoprecipitation assays using FHV-1-infected cell lysate, four groups of immunogenic proteins were identified. MoAbs directed against 60 kDa, 113 kDa, and 143 kDa/108 kDa glycoproteins had virus neutralizing activities, but those against 170 kDa protein did not. Furthermore, MoAbs to the 60 kDa glycoprotein also showed hemagglutination-inhibition activity, indicating that this glycoprotein might be the hemagglutinin.