Non-essential loci in the BamHI-I and -F fragments of the HVT FC126 genome.

The sequence of BamHI-I fragment of the herpesvirus of turkeys (HVT) FC126 strain DNA was analyzed for the presence of potential open reading frames (ORFs). Four complete (ORFs 2 to 5) and 2 partial ORFs (ORFs 1 and 6) were detected. ORFs 2 and 3 were homologous to the HSV-1 UL55 and the EHV-1 gene 3, respectively. The ORF 6 was already partially sequenced by Smith et al. (Virology 207, 205-216, 1995), and was homologous to a Marek's disease virus (MDV) ORF located in a similar position (ORF 21; Ross et al., Virus Genes 7, 33-51, 1993a). No significant homology was found for the other ORFs. ORF 4 was antisense to ORF 3. Two HVT recombinants having an expression cassette inserted into two intergenic sites were generated and tested for viremia in chickens. Results demonstrated that these 2 intergenic loci are non-essential for in vitro and in vivo HVT replication. A 650 bp deletion in the repeat region flanking UL (TRL and IRL (BamHI-F)) has been identified in some DNA molecules of HVT FC126 strain. This deletion covers the entire truncated pp38 homologous ORF and the N-terminus of a small ORF which has no detectable homology with any known gene. Our results indicate that (1) this genomic region including the HVT pp38 homologue was not essential for in vitro and in vivo growth of HVT, and (2) this deletion had no apparent effect on Marek's disease (MD) protection induced by HVT.

Analysis of the biochemical properties of, and complex formation between, glycoproteins H and L of the gamma2 herpesvirus bovine herpesvirus-4.

Genes encoding glycoprotein gH and gL homologues were localized in the genome of the gamma-herpesvirus bovine herpesvirus-4 (BHV-4). Both genes were sequenced and glutathione S-transferase fusion proteins were produced and used to immunize rabbits against the translation products of the two genes. The anti-gH serum recognized a protein with an apparent molecular mass (MM) of 110 kDa both in infected cells and in virions. This protein was sensitive to endo-beta-N-acetylglucosaminase-H (endoH) and endoglycosidase F-N-glycosidase F (endoF-PNGaseF) digestion. A protein with the same relative mobility was immunoprecipitated from infected cells radiolabelled with [3H]glucosamine which confirmed that this product (gp110), now designated BHV-4 gH, was glycosylated. Western blotting with the anti-gL serum detected in infected cells a product with an apparent MM ranging from 31-35 kDa and diffusely migrating protein species ranging from 45-65 kDa. Tunicamycin, monensin, endoH or endoF-PNGaseF treatments showed that both the 31-35 kDa and the 45-65 kDa proteins were glycosylated, gp31-35 being a precursor of the 45-65 kDa glycoprotein species. In radioimmunoprecipitation assays, the anti-gL serum immunoprecipitated from infected cells two glycosylated proteins with apparent MMs of 31-35 kDa (gp31-35) and 45-55 kDa (gp45-55). However a third glycoprotein, gp110, was also immunoprecipitated together with gp31-35 and gp45-55. gp110 and gp45-55 were subsequently confirmed to be virion glycoproteins corresponding to mature forms of BHV-4 gH and gL respectively. In addition, the present study clearly demonstrated complex formation between BHV-4 gH and gL both in virions and in infected cells.

Glycoprotein B of bovine herpesvirus 4 is a major component of the virion, unlike that of two other gammaherpesviruses, Epstein-Barr virus and murine gammaherpesvirus 68.

This study reports that in bovine herpesvirus 4, glycoprotein B (gB) is a heterodimer and a major component of the virion, unlike gBs of Epstein-Barr virus (gp110) and murine gammaherpesvirus 68, two other gammaherpesviruses. These are new characteristics with regard to the general features of gB in the Gammaherpesvirinae subfamily.

Bovine herpesvirus 4: genomic organization and relationship with two other gammaherpesviruses, Epstein-Barr virus and herpesvirus saimiri.

Bovine herpesvirus 4 (BHV-4) belongs to the gammaherpesvirinae subfamily. Although the whole sequence of BHV-4 genome is not known it was possible, based on random sequencing, to assume that its genomic organization consists of genes clustered in blocks whose orientation and location in the genome are conserved within a herpesvirus subfamily. Between these blocks lie genes which are specific to either a particular virus or a virus subfamily. BHV-4 genome consists of 5 gene blocks conserved among the gammaherpesviruses and particularly within the Epstein-Barr virus (EBV) and the herpesvirus saimiri (HVS) genomes. Analysis of the regions located outside the gene blocks showed the presence of 12 open reading frames (ORFs). Protein database comparisons showed that no ORF translation products were similar to proteins encoded by alpha- or beta-herpesviruses. Nevertheless, 5 ORFs were homologous in amino acid sequences to proteins encoded by HVS and one was similar to a protein encoded by both HVS and EBV. On the basis of the molecular data BHV-4 is more closely related to HVS than to EBV. Genes homologous to cellular genes have been described in both HVS and EBV genomes. No genes homologous to presently sequenced cellular genes were found among those found in the BHV-4 genome to date.

Herpesvirus of turkey recombinant viruses expressing infectious bursal disease virus (IBDV) VP2 immunogen induce protection against an IBDV virulent challenge in chickens.

Two recombinant herpesviruses of turkey (HVT) expressing the VP2 protein of infectious bursal disease virus (IBDV or Gumboro disease virus) have been constructed: vHVT001 and vHVT002. The VP2 open reading frame was inserted at the locus of the small subunit of ribonucleotide reductase gene (HSV-1 UL40 homolog) without any exogenous promoter in vHVT001 and at the locus of gl gene (HSV-1 US7 homolog) under the control of the human cytomegalovirus immediate-early promoter in vHVT002. The isolation of these recombinant viruses indicated that the deleted genes were not required for replication of HVT in chicken embryo fibroblasts. Efficacy of these recombinant viruses against IBDV strain 52/70 and Marek's disease virus (MDV strain RB1B) virulent challenges was evaluated in chickens vaccinated at 1 day of age. In the IBDV challenge, a good protection against mortality and bursal gross lesion was observed in vHVT002-vaccinated chickens: 100% with 10(5) PFU dose and 60% with 10(4) PFU dose; in contrast, only a weak level of protection was achieved after vaccination with vHVT001. Protection levels against MDV challenge obtained with vHVT001 and vHVT002 were low (around 10%) compared to that induced by the parental HVT (84%). In spite of the low protection level against MDV, this is the first report which describes induction of full protection against IBDV with a single inoculation of a recombinant virus.

Analysis of bovine herpesvirus 4 genomic regions located outside the conserved gammaherpesvirus gene blocks.

Bovine herpesvirus 4 (BHV-4) DNA sequences located outside the gene blocks conserved among the gammaherpesviruses BHV-4, herpesvirus saimiri (HVS) and Epstein-Barr virus (EBV) were analysed. Twelve potential open reading frames (ORFs) were found. Protein database comparisons showed that no ORF translation products were similar to proteins encoded by alpha- or betaherpesviruses. Nevertheless, six of the ORFs were homologous in amino acid sequences to proteins encoded by HVS but apparently not to those encoded by EBV. Furthermore, the location and orientation of these six ORFs in the BHV-4 genome were similar to the corresponding ORFs in the HVS genome. No genes homologous to known cellular genes were found in the BHV-4 genome; this feature is the major difference between the BHV-4 and HVS genomes with regards to the overall gene content.

Attachment of the gammaherpesvirus bovine herpesvirus 4 is mediated by the interaction of gp8 glycoprotein with heparinlike moieties on the cell surface.

Cell surface heparan sulfate serves as the initial receptor for several alphaherpesviruses and at least one betaherpesvirus. This study shows that during the process of adsorption of the gammaherpesvirus bovine herpesvirus 4 (BHV-4), the viral glycoprotein gp8 interacts with heparinlike moieties of cell surface. This conclusion is based on the following findings. (i) Soluble heparin was capable of blocking BHV-4 infection of Georgia bovine kidney cells by inhibition of viral attachment. (ii) Nevertheless, after virus adsorption to Georgia bovine kidney cells, heparin was partially capable of removing adsorbed virus. (iii) Enzymatic digestion of cell surface heparan sulfate but not of chondroitin sulfates A, B, and C reduced the binding of the virus to the cells, and rendered the cells partially resistant to infection. (iv) Radiolabeled purified BHV-4 bound to wild-type Chinese hamster ovary cells, whereas binding of the virus to mutant Chinese hamster ovary cell lines that where deficient in either all glycosaminoglycans or only heparan sulfate was significantly impaired. (v) Using heparin-affinity chromatography, gp8 glycoprotein was shown to bind specifically to immobilized heparin and to elute in the presence of soluble heparin. These data together showed that the gammaherpesvirus BHV-4, like alphaherpesviruses and one betaherpesvirus, adsorbs to cells by binding to cell surface heparin-like moieties. Therefore, this study extends the group of herpesviruses interacting with heparinlike moieties at the cell surface to a member of the gammaherpesvirinae subfamily.

Molecular biology of bovine herpesvirus type 4.

Bovine herpesvirus type 4 (BHV-4) is a ubiquitous virus of cattle. Its genome is a 144 +/- 6 kb double-stranded DNA consisting of a unique central part (L-DNA) flanked at both ends by tandem repeats called polyrepetitive DNA (prDNA or H-DNA). The overall arrangement of genes has been obtained by the analysis of homologies between short BHV-4 DNA sequences and corresponding genes of Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS). The gene expression is temporally regulated. Glycoprotein precursor p (gp10/gp17) is expressed as gamma 1 polypeptide. Glycoproteins gp1, gp8, gp11 and their precursors are gamma 2 proteins. The analysis of strain variations allows the definition of two types of strains, based on the DNA patterns: the Movar 33/63-like and the DN 599-like strains. Only the M40 strain, isolated in India, fails to fit this classification. The genomic variations have been compiled to build a dendrogram showing three levels of divergence between BHV-4 strains or isolates. The available molecular data indicate that the BHV-4 genome shares much similarity with the DNA of EBV and HVS, two representative members of the gammaherpesvirinae. BHV-4 may therefore be classified in the subfamily gammaherpesvirinae.

Genetic relationships between bovine herpesvirus 4 and the gammaherpesviruses Epstein-Barr virus and herpesvirus saimiri.

The overall arrangement of genes in the unique central part of the bovine herpesvirus type 4 (BHV-4) genome has been deduced by analysis of short DNA sequences. Twenty-three genes conserved in at least one of the completely sequenced herpesviruses have been identified and localized. All of these genes encoded amino acid sequences with higher similarity to proteins of the gammaherpesviruses Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS) than to the homologous products of the alphaherpesviruses varicella-zoster virus and herpes simplex virus type 1 or the betaherpesvirus human cytomegalovirus. The genome organization of BHV-4 had also an overall colinearity with that of the gammaherpesviruses EBV and HVS. Furthermore, the BHV-4 genes content and arrangement were more similar to those of HVS than to those of EBV, suggesting that BHV-4 and HVS are evolutionarily more closely related to each other than either are to EBV. BHV-4 DNA sequences were generally deficient in CpG dinucleotide. This CpG deficiency is characteristic of gammaherpesvirus genomes and suggests that the BHV-4 latent genome is extensively methylated. Despite several biological features similar to those of betaherpesviruses, BHV-4 displays the molecular characteristics of the representative members of the gammaherpesvirinae subfamily.

Location and characterization of the bovine herpesvirus type 4 thymidine kinase gene; comparison with thymidine kinase genes of other herpesviruses.

The location and nucleotide sequence of the bovine herpesvirus type 4 (BHV-4) thymidine kinase (TK) gene was determined. The coding region of the TK gene is 1335 nucleotides long and corresponds to a polypeptide of 445 amino acids. Comparison of TK amino acid sequences of BHV-4 and 16 herpesvirus TKs reveals a greater homology to those of the gammaherpesviruses EBV and specially HVS, than to those of alphaherpesviruses. The open reading frames detected in the vicinity of TK gene were homologous to the corresponding ones in other herpesviruses.

The biology of bovine herpesvirus-4 infection of cattle.

The biology of bovine herpesvirus-4 (BHV-4) infection of cattle is reviewed. The infection is distributed worldwide. Most of isolated viruses are non-pathogenic in cattle; some of them are able to produce a genital disease. Twenty-nine structural polypeptides were described; ten of them are glycosylated. Two major glycoproteins were characterized by monoclonal antibodies. Restriction maps of BHV-4 DNA are available for the enzymes EcoRI, BamHi and HindIII. The strain variations studied by restriction analysis are very weak. The virus is able to persist in a latent state after primary infection. The identified sites of latency are nervous ganglia and mononuclear blood cells. The immune response of cattle after BHV-4 infection is characterized by low or undetectable levels of neutralizing antibodies. Four envelope proteins are recognized by convalescent sera and are the main antigenic components. Skin test remains negative in immunized cattle. Bovine herpesvirus-4 is not strictly species-specific: infection was proved in American bison (Bison bison), African buffalo (Syncerus caffer), sheep and probably cat, because feline herpesvirus-2 is in fact a BHV-4 strain. Finally BHV-4 shares antigenic and genomic relationships with alcelaphine herpesvirus-1, the causal agent of the African form of malignant catarrhal fever.

Experimental infection of bulls with a genital isolate of bovine herpesvirus-4 and reactivation of latent virus with dexamethasone.

Five 13- to 18-month old Belgian Blue bulls were used in this experiment. Four bulls (Nos. 2, 3, 4 and 5) were inoculated intratesticularly with 10(5) plaque-forming units of bovine herpesvirus-4 (BHV-4) in each testicle (Day 0). The challenge BHV-4 strain was previously isolated from testicle cells of a bull exhibiting orchitis and azoospermia. The fifth bull (No. 1) was used as a control and received the same volume of uninfected cell culture supernatant. For 5 days, beginning on Day 51 post-infection, two bulls (Nos. 4 and 5) and the control bull (No. 1) received 0.1 mg kg-1 of dexamethasone. Unilateral castrations were then performed at regular intervals for viral examination. Treatment with dexamethasone reactivated latent BHV-4, but no clinical signs were observed in treated bulls until the end of the experiment (Day 93). Only Bull 3 showed conjunctivitis and temporary azoospermia. The virus was recovered from various samples showing that: (i) BHV-4 can be present in a latent state in the testicles and mononuclear blood cells; (ii) dexamethasone reactivates the virus; (iii) the virus is excreted by nasal and ocular routes. Each infected bull seroconverted and a booster antibody response appeared after dexamethasone treatment as shown by immunofluorescence. Neutralizing antibodies were detected in each bull by complement-dependent neutralization test with titres higher than those obtained by a classical neutralization test. No booster response of neutralizing antibodies was observed after dexamethasone treatment. The antigenically relevant envelope BHV-4 proteins were identified by Western blotting using sera samples from the animals. DNA restriction endonuclease profiles of viruses reisolated after primary infection and reactivation showed only small differences.

Proteins specified by bovine herpesvirus type 4: structural proteins of the virion and identification of two major glycoproteins by using monoclonal antibodies.

Bovine herpesvirus type 4 proteins were identified by PAGE of [35S]methionine- or [3H]glucosamine-labelled purified virions. Thirty-one monoclonal antibodies (MAbs) raised against the V. Test strain were used to identify 29 proteins, ten of which were glycosylated. All of these glycoproteins belonged to the viral envelope and a 140K non-glycosylated protein appeared to be the major nucleocapsid protein. The MAbs were classified into two groups. The first group precipitated three glycoproteins of Mr 150K, 120K and 51K. The 120K and 51K glycoproteins were linked by disulphide bonds and the 150K glycoprotein was linked to the others by non-covalent bonds. The second group precipitated a different 120K glycoprotein.

Production and characterization of monoclonal antibodies to bovid herpesvirus-4.

Thirty-five hybridoma cell lines secreting monoclonal antibodies (Mabs) against bovid herpesvirus-4 (BHV-4) strain V. Test were produced. These hybrid cells resulted from the fusion of SP2/0 myeloma cells with splenocytes of BALB/c mice previously immunized with purified BHV-4. A modified indirect fluorescent antibody test (IFAT) was applied as a screening procedure and was compared with an indirect enzyme-linked immunosorbent assay. The selected Mabs were tested by the same IFAT against a panel of BHV-4 field isolates and against bovine herpesvirus-1, bovine herpesvirus-2 and alcelaphine herpesvirus-1 (AHV-1). Comparison of BHV-4 field isolates with Mabs confirmed their close antigenic relationships, but slight antigenic differences were observed between different isolates. One of the Mabs also reacted against AHV-1, indicating an antigenic relationship between BHV-4 and AHV-1. None of the Mabs reacting with BHV-4 possessed neutralizing activity against the strain used for immunization.