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It was recently reported that canine parvoviruses (CPV) had entered cat populations and induced disease in infected cats, while they had affected only dogs in the past. It is important to determine whether conventional feline panleukopenia virus (FPLV) vaccines protect against recent CPVinfections. In this study, the cross-reactivity of virus-neutralising (VN) and haemagglutinin-inhibition (HI) antibodies in cats induced by FPLVandCPVs were examined. Lower cross-reactivities of VNand HIantibodies against each CPVstrain were observed in cats experimentally inoculated with FPLVor vaccinated with an inactivated FPLVvaccine. In addition, we revealed the existence of a novel type of FPLV, which reacted weakly with antibodies induced by the conventional FPLVvaccine.

The in vivo pathogenicity of canine parvovirus (CPV) type 2c (strain V203) and of CPV type 2a (strain V154) against cats was investigated. Our results indicate that both types of CPV have the potential to induce disease in cats.

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 exression 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.

Summary Sera from dogs which were infected with canine herpesvirus (CHV) were analyzed for their serological reactivities against virus-specific glycoproteins (gps). By sequential immunoblot analysis using sera from experimentally infected dogs, it was found that the antibody response to gp 145/112 appeared first followed by responses to gp 47 and gp 80. In addition, all sera from naturally infected dogs which showed neutralizing activity to CHV reacted with gp 145/112, whereas 77% and 70% reacted with gp 47 and gp 80, respectively. Furthermore, some of the sera also cross-neutralized feline herpesvirus type 1 (FHV-1) and reacted with gp 143/108 of FHV-1, indicating that gp 145/112 of CHV induced cross-neutralizing antibody response to FHV-1.

Summary Thegag gene of a Japanese feline immunodeficiency virus (FIV) isolate, designated as FIV TM 2, was expressed inEscherichia coli as a fusion protein with TrpE. Using this expressed protein, an enzyme-linked immunosorbent assay was developed for detection of antibodies to FIVgag protein in feline sera. With serum samples from a cat experimentally infected with FIV, it was demonstrated that the period of seroconversion detected by this method corresponded to that by Western blotting.

Summary The susceptibility of early-passage feline brain cells and Crandell feline kidney (CRFK) cells to infection with three isolates of feline immunodeficiency virus (FIV) was investigated. The Petaluma strain of FIV could well infect both the feline brain cells and CRFK cells. The KYO-1 strain could well infect the feline brain cells but the replication in CRFK cells was demonstrated only by coculturing fresh feline T-lymphoblastoid cells with the infected cells. On the other hand, the TM1 strain could infect the feline brain cells but not CRFK cells. Moreover, the replicative ability of the TM1 strain in the feline brain cells was much less than the KYO-1 and Petaluma strains. These results indicate that biological differences can be detected among the FIV isolates.

Summary Monoclonal antibodies (MoAbs) against canine herpesvirus (CHV) were produced to identify the immunogenic proteins of the virus carrying neutralizing determinants. A panel of 24 MoAbs showing neutralizing activities was obtained and tentatively classified into 3 different groups based on their reactivity patterns in immunoblotting analysis. Group I consisting of 10 clones was specific for 145/112 kDa; Group II of 9 clones, for 80 kDa; and Group III of 5 clones, for 41 kDa glycoproteins (gps). Complement-requirement for neutralizing activities of the MoAbs suggests that gp 145/112 and gp 80 elicit mainly complement-requiring and -enhanced neutralizing antibodies, while gp 41 elicits complement-independent ones. In addition, these MoAbs were used in ELISA additivity tests for functional and topographical mapping of epitopes in each of the CHV gp. The results indicated that antigenic reactivities of gp 145/112 and gp 80 were, respectively, localized on at least 5 and 7 overlapping epitopes. On the other hand, 4 epitopes were identified on gp 41.

Summary A new collection of eighteen neutralizing monoclonal antibodies (N-MoAbs), raised against feline calicivirus (FCV), was used to analyze neutralizing epitopes of the F 4 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 F 4. 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 F 4 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 F 4 were identified. Immunoblot analysis indicated that four of the seven epitopes existed on the 67 kDa capsid protein of the virus.

Summary The nucleotide sequences of the long terminal repeat of five Japanese, five Argentine and three Australian isolates of feline immunodeficiency virus (FIV) were determined and compared with those of isolates previously described. The results revealed that the Japanese isolates were found to cluster with nucleotide sequence similarity of 95.6%–99.4%. The Australian isolates also clustered with nucleotide sequence similarity of 97.2%–99.4%. The Argentine isolates formed two groups; the LP9 isolate is closely related to the Japanese isolates, whereas the LP1, LP3, LP20 and LP24 isolates are distant from both the Japanese and Australian isolates. From these results, FIV can be divided into three groups, namely: (I) the Californian, Australian and British isolates; (II) the Japanese isolates and one Argentine LP9 isolate; (III) the other Argentine isolates.

To investigate whether the feline CD 4 (fCD 4) molecules are involved in infections of highly lymphotropic feline immunodeficiency virus (FIV) isolates, we expressed fCD 4 stably on Crandell feline kidney cells andFelis catuswhole foetus 4 cells by transfection of a cDNA encoding the fCD 4 glycoprotein, and then infected them with TM 1 and TM 2 strains of FIV, which are unable to infect these cells productively. In spite of fCD 4 being expressed on these cells, no virus production was observed. This result indicates that fCD 4 expression alone cannot induce a productive infection of the FIV TM 1 and TM 2 strains.

Summary We 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.

Summary The gene of Marek's disease virus (MDV) serotype 2 (MDV2) homologous to glycoprotein D (gD) of herpes simplex virus (HSV) was identified and characterized by its nucleotide and predicted amino acid sequences. The MDV2 gD homologous gene contains an open reading frame capable of specifying a polypeptide of 385 amino acids, which include N- and C-terminal hydrophobic domains consistent with signal and anchor regions, respectively, and two potential N-linked glycosylation sites, one of which was located in a highly conserved region when compared to MDV serotype 1 (MDV1) and herpesvirus of turkeys (HVT). By northern blot analysis using a MDV2 gD-specific DNA probe, two highly abundant polycistronic 6.0 and 4.2 kb transcripts were detected in MDV2-infected cells. The genes encoding MDV2 protein kinase (PK), gD, and glycoprotein I (gI) homologues are transcribed to form 3' coterminal mRNAs of 6.0 kb (encoding PK, gD and gI) and 4.2 kb (encoding gD and gI), respectively. By using rapid amplification cDNA end (RACE) method, several RNA start sites, to be thought those of the 4.2 kb mRNA, were detected in the upstream of MDV2 gD homologue.

Summary 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 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.

Summary 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.

Summary A quantitative method for analysis of feline immunodeficiency virus (FIV) was established by the indirect immunofluorescence (IF) test using MYA-1 cells infected with the serially diluted viruses. When we compared this method with the reverse transcriptase (RT) activity assay, the quantitative analysis by the indirect IF test was more sensitive than that by RT activity assay. The relationship between TCID50 measured by the indirect IF test and RT activity was linear. However, the RT activity assay was not suitable for measurement of low amounts of FIV in the supernatants of FIV-infected cultures.

Glycoproteins B (MDV1-gB) and C (MDV1-gC) of Marek's 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.

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 anEcoRI 6.6 kbp fragment. In addition, a partial UL 21 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 aN-terminal hydrophobic signal sequence,C-terminal transmembrane domain, and putativeN-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.