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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.

In order to confirm the in vivo effectiveness of anti- feline herpesvirus type 1 (FHV-1) mouse-cat chimeric antibody (FJH2), and anti-feline calicivirus (FCV) mouse-cat chimeric antibody (F1D7), cats that had been experimentally infected with FHV-1 or FCV were administered intravenously with the chimeric antibodies, and observed for clinical manifestations. The symptoms due to FHV-1 or FCV infection in the cats administered FJH2 or F1D7 were obviously decreased when compared with those of the non-administered control cats. From these results, it was confirmed that both FJH2 and F1D7 were effective at reducing the appearance of symptoms due to FHV-1 and FCV infection, respectively.

In order to investigate whether a recombinant viral vaccine composed of a feline herpesvirus type 1 (FHVI) vector and an immunogenic antigen of Toxoplasma gondii can induce a protective immunity in cats, a recombinant FHV1 expressing ROP2 antigen of T. gondii was prepared. We introduced a DNA fragment encoding 1-538 amino acid residues of ROP2 precursor into FHV1 genome under the control of a cytomegalovirus promoter. The recombinant FHV1 (FHV/ROP2) successfully expressed a 59 kDa antigen that was recognized by anti-ROP2 antibodies. Vaccination of cats with FHV/ROP2 induced serum IgG recognizing the native antigen. Moreover the antibodies inhibited the in vitro invasion of tachyzoites. The vaccination also accelerated the IgG response after T. gondii infection and reduced brain parasite load in cats.

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.

We previously reported the attenuation of thymidine kinase (TK) deficient mutant (C7301dlTK) of feline herpesvirus type 1 (FHV-1) in cats and the construction of a recombinant FHV-1 (C7301dlTK-Cap) inserted a precursor capsid gene of feline calcivirus (FCV) into the TK deletion locus of the C7301dlTK. In this study, we constructed a further improved recombinant FHV-1 (dlTK(gCp)-Cap) carrying a putative FHV-1 gC promoter sequence upstream of the FCV precursor capsid gene of the C7301dlTK-Cap. Growth kinetics of the dlTK(gCp)-Cap in cell cultures was similar to those of C7301dlTK and C7301dlTK-Cap. A strong expression of FCV immunogenic antigen by dlTK(gCp)-Cap was confirmed by indirect immunofluorescence and enzyme-linked immunosorbent assays. In addition, one vaccination with dlTK(gCp)-Cap protected cats more effective against subsequent virulent FCV challenge than that with C7301dlTK-Cap.

We investigated the possibility of in vitro recombination of three different strains 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.

The effects of treatment with chemical agents or infection with feline viruses on protein-binding properties of the feline immunodeficiency virus (FIV) long terminal repeat (LTR) were examined by gel-mobility-shift assays using oligonucleotides designed to represent putative AP-1 or ATF motif from the FIV LTR. Infection with FIV led to less nuclear proteins binding to the AP-1 and ATF sites, suggesting that proteins binding to the sites were consumed or suppressed by FIV-replication in FIV-infected cells. Nuclear proteins that bind to the AP-1 or ATF site were examined by using extracts from Crandell feline kidney (CRFK) cells treated with TPA (a phorbol ester; a strong activator of protein kinase C) or forskolin (an inducer of cyclic-AMP), or infection with feline herpesvirus type 1 (FHV-1). Although TPA or forskolin treatment moderately increased the level of both proteins that bound to AP-1 and ATF sites, FHV-1 infection markedly changed the protein-binding patterns of the sites. Furthermore, FHV-1-induced proteins that bind adjacent to the transcriptional initiation site of FIV promoter were also observed in FHV-1-infected CRFK cells, suggesting that the FHV-1-induced-proteins affects the transcription of FIV through the AP-1, ATF and leader sequences.

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.

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.

Human and animal herpesviruses are able to stimulate gene expression of various human and animal retroviruses and the activation appears to be a common theme. It is of interest why the retroviruses can be commonly activated by herpesviruses despite clear diversities in each mechanism for retrovirus gene expression. In this review, three typical examples of the interactions; human immunodeficiency virus and human herpesviruses, avian retroviruses and Marek's disease virus, and feline immunodeficiency virus and feline herpesvirus type I will be given to respond the above question on the basis of the comparative study of molecular interactions.

The genomic heterogeneity of 78 isolates of feline herpesvirus type 1 (FHV-1) recently isolated from cats suspected to have feline viral rhinotracheitis was analyzed by examining the digestion patterns found with restriction endonuclease MluI. The FHV-1 field isolates were classified into at least three genotypes, namely, the C7301, F2 (an attenuated vaccine strain), and C7805 types. The C7301 type seems to be a major type, since 64 of the 78 isolates belonged to this type. Eight and six isolates belonged to the F2 and C7805 types, respectively. Compared with the C7301 type, the heterogeneous region of the F2 type was localized to a 4.3-kbp EcoRI fragment within the US segment and the heterogeneous region of the C7805 type was localized to a 5.5-kbp XbaI fragment within the UL segment. Northern (RNA) blot analysis revealed no differences in the products transcribed from these regions. In addition, nucleotide sequence analysis showed that the MluI sites not found in the F2 and C7805 types were located in the regions homologous to the herpes simplex virus type 1 gI and UL5 genes, respectively.

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.

We constructed a series of deletion mutants of feline immunodeficiency virus (FIV) long terminal repeat (LTR) to identify the regions that regulate gene expression and are responsive to trans-activation of FIV LTR by feline herpes-virus type 1 (FHV-1). We demonstrated that sequences between -124 and -79, and between -21 and -32 (relative to the cap site) are essential for gene expression of FIV in Felis catus whole fetus 4 (fcwf-4) cells. Further, we demonstrated that the sequence between -63 and -23 responds to trans-activation of FIV LTR by FHV-1 in fcwf-4 cells.

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.

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.

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.

Various strains immunologically related to Marek's disease virus (MDV) have been subdivided into three serotypes: serotype 1, pathogenic strains of MDV and attenuated or apathogenic variants derived from them; serotype 2, naturally occurring apathogenic strains of MDV; serotype 3, herpesvirus of turkey (HVT). The viral genome structures of these three serotypes were compared by a simple, practical method using total DNA extracted from virus-infected cells instead of viral DNA purified from virions. The restriction endonuclease-cleavage patterns of serotype 2 viral DNA were found to differ from those of either serotype 1 MDV or serotype 3. Under stringent conditions, no significant DNA homology was detected among the three serotype viruses, except in a restricted portion of these viral genomes. Northern blot hybridization experiments suggested that virus-specific polyadenylated RNA of about 2.4 kilobases was transcribed from a restricted portion showing close homology in these viruses. Southern blot hybridization under less stringent conditions revealed that regions with weak homology were distributed over most of the viral genomes of the three serotypes. Two types of virus-specific glycoproteins, gA and gB, were identified in the immunoprecipitates of the culture medium and cell lysates, respectively, of serotype 2-infected cultures with monoclonal antibodies or hyperimmune antisera cross reactive with serotype 1 MDV and serotype 3 HVT, and detected on the surface of serotype 2-infected cells by the membrane immunofluorescence test. These results indicate a close evolutionary relationship among these three viral serotypes.

An agar-gel precipitation antigen prepared from the skin (feather-Ag) of chicken infected with JM strain of Marek's disease herpesvirus (MDHV) and cell extracts of cultures infected with either Type 2 plaque producing agent (PPA) of MDHV or turkey herpesvirus (HVT) (Type 2-Ag, HVT-Ag) had 1 precipitation line in common. Hyperimmune sera to the common antigen (common-Ag) neutralized to a greater extent by homologous antiserum. Absorption of HVT antiserum or hyperimmune serum to Type 2 PPA with common-Ag reduced their neutralizing activity. This reduction was almost complete with homologous antigen but less complete with heterologous antigen. The location of antigen in Type 2 PPA and HVT infected cells was determined using hyperimmune serum to the common-Ag in fluorescent antibody tests. Antigen mainly occurred in the cytoplasm of cells corresponding to the rounded refractile cells in the plaques, whereas unfixed cells showed antigen on the cytoplasmic membrane. The common-Ag associated with MDHV or HVT infections might be a virus structural component which is associated with the envelope.

The appearance of two kinds of membrane antigen (MA) in Marek's disease herpesvirus (MDHV) or herpesvirus of turkey (HVT) infected cells was examined using antiserum to the common antigen (common-Ag). No early-appearing MA(EMA) was detected in these cultures until 16 hours post-inoculation, after which a number of cells in these cultures had late-appearing membrane antigen (LMA) as detected by immunofluorescence (IF). Fluids from cultures infected with these viruses were examined for the presence of the common-Ag by agar-gel precipitation (AGP) test. No common-Ag was detected until 16 hours, but after 24 hours, it was detected by AGP test. These results suggest that the common-Ag is related to LMA but not to EMA.

Three-dimensional structures of the core of Marek's disease virus and herpesvirus of turkey were examined by the tilting apparatus of an electron microscope. Various types of the core found in the infected cells were considered to represent developmental stages of the viruses. The basic structure of the core consisted of a toroid surrounding a cylindrical mass, which was clearly demonstrated by tilting the core in two directions. A cylindrical mass spooled by more than two toroids, which seemed to constitute a spiral band of 10 to 20 nm, was demonstrated. The maturation process of the cores of the viruses was also discussed.

In a study of outbreaks of Marek's disease in quails, 220 adult quails vaccinated with herpesvirus of turkeys (HVT) were examined pathologically and serologically for Marek's disease (MD). Forty-three of the 220 quails exhibited microscopic lesions similar to those of chickens with MD. MD-virus-specific antigen was found in feather tips of 44 of the 220 birds, and the HVT-specific antibody was found in sera of 56 of the 220 birds by agar gel precipitation. There was a positive correlation between the incidence of lymphomatous changes and the presence of MD-virus-specific antigen, and there was a negative correlation between the incidence of lymphomatous changes and the presence of antibody against HVT on a flock basis.

The relation between the neutralization of herpesvirus of turkeys HVT) and the antibodies to early appearing membrane antigen (EMA), late appearing membrane antigen (LMA) and intracellular antigen (IA) induced by the virus was examined by blocking of direct immunofluorescence using 38 sera from chickens infected with HVT. Our results provide evidence that anti-LMA is significant in the neutralization of the infectivity (P less than 0.001) since the neutralizing capacity of the serum was related to its blocking index (BI) and not to its anti-EMA titre (BI). Furthermore, the anti-IA titres (BI) of the sera were also related to their neutralizing activity to a lesser extent (P less than 0.025). However, no relationships among any two of the titres of EMA, LMA and IA were observed. The implication of these results of the relationship of LMA to infectious virus particles is discussed.

Quail embryo fibroblasts were used to investigate how rabbit and chicken antisera against chicken erythrocytes carrying different B alleles of the major histocompatibility antigens affect the neutralization of herpesvirus of turkeys (HVT). Although the neutralizing activities of these antisera were rather low, the HVT propagated in chicken embryo fibroblasts (CEF) from certain genotypic embryos was neutralized more by the antisera to the corresponding erythrocytes. After absorption of these antisera with homologous erythrocytes, the neutralizing activity of the absorbed sera was reduced slightly. These results reveal that the virion antigens of HVT might be partially associated with the host cell antigens of the fibroblast infected with the virus. The virus grown in these cells might incorporate the host cell antigens, including histocompatibility antigens, into the virion envelope.

A variant turkey herpesvirus (HVT/VT) which releases 10(5.0) plaque-forming units per 0.1 ml of cell-free virus into the culture medium was established from the prototype HVT FC 126 strain (HVT/WT). Many enveloped and naked virions of HVT/VT but only naked virions of HVT/WT were found in the culture medium. By the HindIII restriction cleavage patterns, the HVT/VT DNAs were almost identical to those of HVT/WT. The HVT/VT was replication-defective in chickens.

Blocking of the direct immunofluorescence (IF) reaction allowed titration of anti-membrane antigen (MA) or anti-intracellular antigen (IA) antibodies induced by herpesvirus of turkeys (HVT). The titers (blocking index) obtained by the blocking IF test were well correlated with those determined by the IF test. The results suggest that the blocking IF test was a simpler and better method for titration of a large number of serum samples from chickens infected with HVT.

The difference between intracellular antigen (IA) and membrane antigen (MA) induced by herpesvirus of turkeys (HVT) was examined by immunofluorescence analysis. Convalescent sera from chickens infected with HVT (HVT convalescent serum) and hyperimmune sera from chickens immunized with partially pruified virus (anti-HVT particle serum) or with a major HVT soluble precipitin antigen (anti-common Ag serum) were used as the source of antisera to specify both antigens in these experiments. Absorption of a convalescent serum from HVT-inoculated chicken with IA positive cells, resulted in reduction of their reactivity to IA but not to MA. Differential absorption of the hyperimmune sera by IA positive cells and MA positive cells resulted in reduction of their reactivity to homologous cells but not to heterlogous cells which were used in the absorptions. When the relationship between the levels of anti-IA and anti-MA antibodies was examined by a blocking immunofluorescence test using 38 sera from chickens inoculated with HVT, no correlation was observed (p greater than 0.25). These results provide evidence that MA and IA are antigenically different from each other.

Serological and genetical relationships of three salmonid herpesviruses [strain H-83, OO-7812 strain of Oncorhynchus masou virus (OMV) and Herpesvirus salmonis] were investigated. Although the serological relationships of these viruses were not distinctly demonstrated by cross-neutralization test, cross enzyme-linked immunosorbent assay revealed that strain H-83 and OMV appeared to be serologically more closely related to each other than to H. salmonis. Strain H-83 was genetically similar to OMV but distinct from H. salmonis by restriction endonuclease cleavage analysis and DNA-DNA hybridization test. These results indicated that strain H-83 should be classified under the same group as OMV, and H. salmonis belong to another group of salmonid herpesviruses.

The serological and biological properties of the type 2 plaque-producing agent (PPA) derived from the Cal-1 strain of Marek's disease virus (MDV) were compared with those of reference strains of the three serotypes of MDV and herpesvirus of turkeys (HVT) groups; namely JM, HPRS-24 strains of MDV and the FC-126 strain of HVT for serotype 1, 2, and 3, respectively. By agar gel precipitation (AGP), indirect fluorescent antibody and virus neutralization tests, type 2 PPA was related but not identical to the FC-126 strain. By the AGP test, type 2 PPA showed a poor ability to synthesize B antigen and the A antigen was different from that of strain FC-126. To compare the virological characteristics of type 2 PPA with the reference strains, the release of cell-free virus into supernatants of infected cell cultures and titers of cell-free virus extracted sonically from infected cell cultures were examined. Cell-free type 2 PPA virus was easily detected in the supernatants and extracted from infected cell cultures. These properties were similar to reference strains of serotype 2 and 3. Next, the structural similarities of viral DNAs of type 2 PPA and strain FC-126 were examined by Southern blot hybridization. The restriction endonuclease-cleavage patterns of DNA of type 2 PPA were very similar but not identical to those of the FC-126 strain. In chickens inoculated with type 2 PPA, splenic lymphocytes supported a non-productive latent infection as did also those from chickens inoculated with the FC-126 or HPRS-24 strains. From these results, type 2 PPA appears to belong to serotype 3 of MDV and HVT groups. The origin of type 2 PPA is discussed.

Serial passages in culture of herpesvirus of turkeys resulted in structural genomic changes at the regions common to two virus strains with loss of their protective ability against Marek's disease.