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Feline herpesvirus type 1 (FHV-1) is a causative agent of feline viral rhinotracheitis and belongs to the subfamily Alphaherpesvirinae of the family Herpesviridae. Since first isolated in 1958 by Crandell and Maurer, FHV-1 is distributed worldwide and is the most clinically significant agent for respiratory infections in cats. In this review, we describe the recent findings with properties and functions of FHV-1 glycoproteins, especially hemagglutinins.

The nucleotide sequence of the glycoprotein B (gB) homologous gene of feline herpesvirus type 1 (FHV-1) was determined. The gene was found to be located within a 9.6 kbp SalI fragment by Southern-blot hybridization with a probe derived from the herpes simplex virus type 1 (HSV-1) gB DNA sequence. Furthermore, the predominant portion of the coding sequences was mapped to a 1.9 kbp Hin cII-EcoRI and its flanking 2.7 kbp Eco RI-Eco RI subfragments in the 9.6 kbp SalI fragment. The entire nucleotide sequence revealed that the FHV-1 gB homologous gene is capable of encoding a polypeptide of 948 amino acids. The predicted precursor polypeptide derived from this open reading frame could have a calculated M(r) of 106 kDa in unglycosylated form and contains ten potential N-linked glycosylation sites and a probable internal proteolytic cleavage site. By Northern-blot analysis using portions of the open reading frame as a probe, 3.9 and 3.3 kb RNA transcripts were identified in FHV-1 infected cells. The alignment of the amino acid sequence of the FHV-1 gB homologue with those of 14 other herpesviruses revealed that 10 cysteine residues were completely conserved. Meanwhile, when evolutionary trees were generated among these herpesvirus gB counterparts, the FHV-1 gB homologous nucleotide sequence seems to be closely related to equine herpesvirus type 4 and its amino acid sequence to pseudorabies virus.

Heterogeneity of 9 feline herpesvirus type 1 (FHV-1) strains consisting of the prototype C27 strain, one French isolate, six Japanese isolates, and the attenuated vaccine F2 strain was examined by biological, immunological, and molecular biological methods. No significant difference was observed in virus growth and antigenic properties among the strains in Crandell feline kidney cell cultures. Hemagglutination activity was also detected in all extracts of cells infected with each strain. However, in immunoblot analysis, a virus-structural immunogenic protein with an M(r) of 36 kDa was lacking in 2 strains, one of which was the vaccine F2 strain, whereas the other immunogenic proteins including three kinds of major glycoproteins were detected in all strains without differences in electrophoretic mobilities. Furthermore, when restriction endonuclease analysis was performed to examine the genomic heterogeneity of strains, the cleavage patterns with the enzyme MluI showed a genomic heterogeneity between wild and vaccine strains. In contrast, only a slight variation in the sizes of some fragments was shown with most of the 7 other enzymes used. These results indicated that the lack of the 36 kDa protein and the MluI cleavage pattern could be used as markers of the vaccine F2 strain. The specific markers are important not only to control the quality of the vaccine but also to evaluate the vaccine immunity in FHV-1 infection in cats.

By transfection of a recombinant plasmid containing the feline immunodeficiency virus (FIV) long terminal repeat (LTR) linked to the chloramphenicol acetyltransferase (CAT) gene followed by infection of feline herpesvirus type 1 (FHV-1) into Crandell feline kidney cells and Felis catus whole fetus 4 cells, enhancement of CAT activity was demonstrated. Furthermore, individual feline T-lymphocytes were productively co-infected with both FIV and FHV-1 in vitro as determined by two-color immunofluorescence and electron microscopy analyses. These results revealed the transactivation of the FIV LTR by FHV-1 and the dual infection of T-lymphocytes with both viruses. The possibility that FHV-1 might be a cofactor which plays a role in the pathogenesis of FIV infection is discussed.

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) were used to characterize feline herpesvirus type 1 (FHV-1) glycoproteins (gp). Intracellular localization and transport of these proteins as revealed by a sequential indirect immunofluorescence assay (IFA) on fixed infected cells showed slight differences between FHV-1 gp143/108 and gp113. Antibodies against gp143/108 first showed membrane fluorescence at 4 hrs post-infection (PI) followed by a pronounced perinuclear and cytoplasmic staining from 8 hrs PI onwards. Those reacting with gp113 showed the same pattern but fluorescence did not appear until 8 hrs PI. In contrast, MoAbs against gp60 first showed para- and perinuclear staining at 12 hrs PI which became intranuclear at 16 hrs PI, followed by intracytoplasmic staining at 20 hrs PI. Sequential IFA of unfixed infected cells revealed that the three glycoproteins were expressed on the cell surface membrane as well. Topographical mapping of the functional epitopes of gp113 by ELISA additivity test indicated the presence of 2 antigenic domains--a neutralizing domain consisting of 3 overlapping epitopes and a non-neutralizing domain. On the other hand, gp143/108 contained only one antigenic site consisting of 5 similar or overlapping epitopes, one of which seemed to be a conserved region recognized by all MoAbs reacting to this protein.

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.

Polyvalent and monoclonal antibodies were used to demonstrate serological and antigenic relationships between feline herpesvirus type 1 (FHV-1) and canine herpesvirus (CHV). Using virus neutralization tests, enzyme-linked immunosorbent and indirect immunofluorescence assays, and immunoblotting analysis, reciprocal cross-reactivities to the heterologous virus were observed with some polyvalent and monoclonal antibodies. One monoclonal antibody against CHV neutralized FHV-1 infectivity and one monoclonal antibody against FHV-1 inhibited the hemagglutination activity of CHV as well as FHV-1-infected mouse serum. The major cross-reacting proteins were identified as the 143/108 kDa and 60 kDa glycoproteins of FHV-1 and the 145/112 kDa and 41 kDa glycoproteins of CHV. Previously, we have identified the 60 kDa and 41 kDa glycoproteins as the hemagglutinins of FHV-1 and CHV, respectively. The present results indicated the presence of shared antigenic determinants among these proteins.

The crude hemagglutinin of feline herpesvirus type 1 (FHV-1), solubilized from infected fcwf-4 cells by detergents, was partially purified by three kinds of chromatographic methods. Lectin-affinity chromatography showed the hemagglutination (HA) activity in fractions, which was bound to Concanavalin A-sepharose and then eluted by alpha-methyl D-mannoside, suggesting that the hemagglutinin might include a glycoprotein. Ion-exchange and gel-exclusion chromatographies were also capable of purifying the detergent-soluble crude hemagglutinin. When peak HA fractions, which were obtained from each of the three procedures, were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the gel-exclusion chromatography was the most effective method. Electrophoreic analysis also showed only one band of 59,000 (59K) molecular weight protein, which was commonly observed in the three partially purified hemagglutinins with silver staining. In addition, the 59K protein band was clearly recognized in immunoblot analysis of the infected cell lysates using infected cat serum. These observations suggest that the FHV-1 detergent-soluble hemagglutinin from infected fcwf-4 cells may be closely related to a 59K immunogenic glycoprotein.

Hemagglutination (HA) activity of feline herpesvirus type 1 (FHV-1) propagated in feline lung cell culture and two established feline cell lines, CRFK and fcwf-4, was investigated. Intra- and extracellular crude samples obtained from those infected cell cultures did not show HA activity. However, when treated with tween 80-ether, HA activity appeared. There was no correlation between virus infectivity titers and the HA titers at various harvesting times, and besides, hemagglutinins were found in intracellular samples at the early stage of infection. By ultrasonic destruction of the infected fcwf-4 cells, high titer hemagglutinins were obtained. High titer hemagglutinins were also extracted successfully from infected fcwf-4 cell membranes by solubilization with any of the three detergents: Triton X-100, DOC, and CHAPS. The optimal concentrations of each detergent for solubilizing hemagglutinin were 0.05 (v/v)%, 0.5 (w/v)%, and 0.1-0.2 (w/v)%, respectively. The HA activities of both the ultrasonic-treated hemagglutinin and the detergent-soluble hemagglutinin from infected fcwf-4 cells were inhibited specifically by anti-FHV-1 sera. Therefore, either hemagglutinin could be used as HA antigen for the hemagglutination-inhibition test.