A live attenuated glycoprotein E negative bovine herpesvirus 1 vaccine induces a partial cross-protection against caprine herpesvirus 1 infection in goats

Taking into account the close antigenic relationship between bovine herpesvirus 1 (BoHV-1) and caprine herpesvirus 1 (CpHV-1), a live attenuated glycoprotein E (gE) negative BoHV-1 vaccine was assessed in goats with the aim to protect against CpHV-1 infection. Vaccine safety was evaluated by intranasal inoculation of two groups of goats with either a gE-negative BoHV-1 vaccine or a virulent BoHV-1. The length of viral excretion and the peak viral titre were reduced with the gE-negative vaccine. To assess the efficacy, two goats were inoculated intranasally twice 2 weeks apart with a gE-negative BoHV-1 vaccine. Four weeks later, immunised and control goats were challenged with CpHV-1. A 2 log(10) reduction in the peak viral titre was observed and the challenge virus excretion lasted 2 days more in immunised than in control goats. These data indicate the safety and the partial efficacy of a live attenuated gE-negative BoHV-1 vaccine intranasally administrated in goats.     

Construction and characterization of a glycoprotein E gene-deleted bovine herpesvirus type 1 recombinant

OBJECTIVE: To construct and characterize a recombinant glycoprotein (g)E gene-deleted bovine herpesvirus (BHV) type 1 (BHV-1). PROCEDURE: The BHV-1 gEgene-coding region and the flanking upstream and downstream sequences were cloned. The aforementioned cloned DNA was digested with suitable enzymes to release the amino terminal two thirds of that region, and was ligated to the beta-galactosidase (beta-gal) gene. The resulting plasmid DNA was cotransfected with DNA from full-length, wild-type (WT), BHV-1 Cooper strain of the virus. Recombinant viruses expressing beta-gal (blue plaques) were plaque purified and assayed further by blot hybridization for genetic characterization and by immunoblotting for reactivity against BHV-1 gE peptide-specific rabbit polyclonal antibody. One recombinant virus, gEdelta3.1IBR, was characterized in vitro and in vivo. The ability of the recombinant virus to induce BHV-1 neutralizing antibodies in infected calves was investigated by plaque-reduction tests. RESULTS AND CONCLUSIONS: The gEdelta3.1IBR virus contained a deletion in the viral gE gene-coding sequences where a stable chimeric reporter (beta-gal) gene was inserted. One-step growth kinetics and virus yield of the recombinant and parent viruses were similar, but early after infection, the recombinant virus yield was comparatively less. After intranasal inoculation, the recombinant gEdelta3.1IBR virus replicated in the upper respiratory tract of calves, but the amount of progeny viruses produced was hundredsfold reduced, and duration of virus shedding was shorter. Results of in vivo calf experiments and serum neutralization tests indicated that deleting the gE gene has little effect on inducing neutralizing antibodies against BHV-1, but is sufficient to reduce BHV-1 virulence in calves.     

Antibody response to glycoprotein E after bovine herpesvirus type 1 infection in passively immunised, glycoprotein E-negative calves

This study was conducted to determine whether young calves with maternal antibodies against bovine herpesvirus type 1 (BHV-1) but without antibodies against glycoprotein E (gE) can produce an active antibody response to gE after a BHV-1 infection. Five calves received at birth colostrum from gE-seronegative cows which had been vaccinated two or three times with an inactivated BHV-1, gE-deleted marker vaccine. After inoculation with a wild-type virulent strain of BHV-1, all the passively immunised gE-negative calves shed virus in large amounts in their nasal secretions. All the calves seroconverted to gE within two to four weeks after inoculation and then had high levels of gE antibodies for at least four months. The development of an active cell-mediated immune response was also detected by in vitro BHV-1-specific interferon-gamma assays. All the calves were latently infected, because one of them re-excreted the virus spontaneously and the other four did so after being treated with dexamethasone. The results showed that under the conditions of this work the gE-negative marker could also distinguish between passively immunised and latently infected calves.     

Protein display by bovine herpesvirus type 1 glycoprotein B

Glycoprotein B (gB) of bovine herpesvirus 1 (BHV-1), a major component of the viral envelope, is essential for membrane fusion during entry and cell-to-cell spread. It is cleaved in the trans-Golgi network by the proprotein convertase furin. Integration of the open reading frame (ORF) encoding a mutated gB with a second furin cleavage site and mature boIFN-α as intervening peptide between the amino-terminal (NH₂) and carboxy-terminal (COOH) gB subunits yielded recombinant BHV-1/gB2FuIFN-α which, unexpectedly, express gB with an enlarged NH₂-subunit of 90 kDa. Here we show that boIFN-α-specific antibodies bind to the 90 kDa gB subunit and efficiently neutralize BHV-1/gB2FuIN-α infectivity. We also show that inactivated BHV-1/gB2FuIN-α virions induce an antiviral state in cells incubated with UV-inactivated particles. These results demonstrate that the 90 kDa protein is a NH₂-subunit/boIFN-α fusion protein whose boIFN-α domain is biologically active. To verify that BHV-1 gB is suitable for the display of (glyco)proteins on the surface of virions we constructed BHV-1 recombinants expressing within gB the first 273 amino acids of the NH₂-subunit (HA1) of avian influenza haemagglutinin, either flanked by two furin cleavage sites or with only one cleavage site between a gB/NH₂_HA1 fusion protein and the COOH subunit. The resulting recombinant BHV-1/gB2FuHA1 expressed gB from which 55 kDa HA1 was excised and secreted. In contrast, gB from BHV-1/gB_NH₂HA1 infected cells retained HA1 as fusion protein with the NH₂-subunit. Immunoblotting and neutralization analyses revealed that HA1 is incorporated into the envelope BHV-1/gB/NH₂_HA1 particles and exposed to the exterior of virions. Thus, this novel approach enables display of polypeptides and (glyco)proteins of at least 273 amino acids on viral particles which is of particular interest for development of novel diagnostics and vaccines as well as for, e.g. gene therapy applications especially when biologically active ligands need to be presented.     

A specific PCR to differentiate between gE negative vaccine and wildtype bovine herpesvirus type 1 strains

In the context of infectious bovine rhinotracheitis (IBR) control programmes using glycoprotein E (gE) deleted marker vaccines, a PCR assay was developed to allow the genotypic differentiation between wildtype bovine herpesvirus type 1 (BoHV-1) and gE negative strains. This assay is based on the PCR amplification of a 281 bp DNA fragment within the gE gene. The specificity of the amplification was confirmed by restriction endonuclease analysis and nucleotide sequencing of the PCR product. Its ability to determine the gE genotype of BoHV-1 strains was demonstrated on isolates coming from 20 experimental calves infected with four different BoHV-1 strains. This PCR assay may be a useful tool for monitoring the spread of live marker vaccine and the gE genotype of viral field isolates.     

Isolation of a glycoprotein E-deleted bovine herpesvirus type 1 strain in the field

During a field trial to evaluate the efficacy of repeated vaccinations with bovine herpesvirus type 1 (BHV-1) marker vaccines, a glycoprotein E (gE)-negative BHV-1 strain was isolated from the nasal secretions of two cows, eight months after vaccination with a gE-negative live-attenuated vaccine, initially given intranasally, then intramuscularly. The strain isolated was characterised using immunofluorescence, restriction analysis and PCR. All the techniques used identified the isolated virus as a gE-negative BHV-1 phenotypically and genotypically identical to the Za strain used as a control.     

Molecular cloning and characterization of bovine P-selectin glycoprotein ligand-1

Human P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin expressed on leukocytes that binds selectins. Here, we report that the open reading frame (ORF) of bovine PSGL-1 (bPSGL-1) cDNA is 1284 base pairs in length, predicting a protein of 427 amino acids including an 18-amino-acid signal peptide, an extracellular region with a mucin-like domain, and transmembrane and cytoplasmic domains. The amino acid sequence of bPSGL-1 demonstrated 52, 49 and 40% overall homology to equine, human and mouse, respectively. A single extracellular cysteine, at the transmembrane and extracellular domain junction, suggests a disulfide-bonding pattern. Alignment of bovine with equine, human and mouse PSGL-1 demonstrates high conservation of transmembrane and cytoplasmic domains, but diversity of the extracellular domain, especially in the anionic NH(2)-terminal of PSGL-1, the putative P-selectin binding domain. In the NH(2)-terminal of bPSGL-1, there are three potential tyrosine sulfation sites and three potential threonine O-glycosylation sites, all of which are required for P-selectin binding in human PSGL-1 (hPSGL-1). bPSGL-1 shares only 57% homology in amino acid sequence with the corresponding epitope region which binds the monoclonal antibody PL1 for hPSGL-1, and no cross-reactivity was found in bovine leukocytes. In summary, bPSGL-1 shares homology with hPSGl-1, but has differences in the putative extracellular P-selectin binding domain.     

A combined bovine herpesvirus 1 gB-gD DNA vaccine induces immune response in mice

Although DNA vaccines have several advantages over conventional vaccines, antibody production and protection are often not adequate, particularly in single plasmid vaccine formulations. Here we assessed the potential for a combined vaccine based on plasmids encoding the membrane-anchored or secreted forms of bovine herpesvirus type 1 (BHV-1) glycoprotein B and D (gB and gD) to induce neutralizing and cell mediated immune responses in mice. Animals were injected by intramuscular, subcutaneous and intranasal routes. Mice immunized with the combined vaccine containing the secreted forms of BHV-1 glycoproteins developed higher titers of anti-BHV-1 neutralizing antibodies, compared to wild type gB/gD combined plasmids and to single plasmid injected groups. Cellular immunity was also developed in mice immunized with combined vaccines, whereas low or no response were observed in single plasmid injected animals. The data suggest the potential use of this combined vaccine in in vivo trials of calves, in order to evaluate its protective efficacy.     

Antibodies against bovine herpesvirus (BHV) 5 may be differentiated from antibodies against BHV1 in a BHV1 glycoprotein E blocking ELISA

We examined whether antibodies against bovine herpesvirus (BHV) 5 cross-react with BHV1 antigens and whether they could interfere with BHV1 eradication programmes. Six calves were experimentally infected with different doses of BHV5 strain N569; homologous antibodies were first detectable on day 11 post infection; they cross-reacted in a BHV1 virus neutralisation test, in a BHV1-glycoprotein (g)-B blocking ELISA and in a BHV1-gE ELISA, but not in a BHV1-gE blocking ELISA. This study indicates that, in ongoing BHV1 eradication programmes, based on vaccines that lack gE, BHV5 infections may not lead to false-positive serological reactions in case cattle are tested for BHV1-gE antibodies by the BHV1-gE blocking ELISA; antibodies against BHV5 may be differentiated from antibodies against BHV1. The BHV1-gE blocking ELISA may, therefore, offer opportunities for the serological differentiation between BHV1 and BHV5 infections.     

Recombination in the alphaherpesvirus bovine herpesvirus 1

Herpesviruses are DNA viruses characterized by a low rate of nucleotide substitution. Therefore, other mechanisms must be involved to their evolution, like recombination that can be seen as an essential evolutionary driving force of these viruses. Recombination contributes to the long-term evolution of alphaherpesviruses. It acts also to continuously create new alphaherpesvirus strains. We have used bovine herpesvirus 1 to investigate recombination both within DNA concatemers in infected cells and in vitro and in vivo at the end of the lytic cycle. The following results have been obtained: (i) intramolecular recombination occurs at the level of concatemers and gives rise to genomic segment inversions; (ii) intraspecific recombination occurs frequently both in vitro and in vivo; (iii) interspecific recombination is possible and requires two highly genetically related viruses; (iv) only simultaneous or closely separated infections lead to the production of recombinant viruses; (v) recombination between wild-type and glycoprotein defective vaccine virus can produce a glycoprotein defective virus keeping part of the virulence of parental wild-type virus. Recombination, by exchanging genomic segments, may modify the virulence of alphaherpesviruses. It must be carefully assessed for the biosafety of antiviral therapy, alphaherpesvirus-based vectors and live attenuated vaccines.     

Bovine herpesvirus 5 BICP0 complements the bovine herpesvirus 1 homolog

Bovine herpesvirus 1 (BoHV-1) and BoHV-5 are closely related (82% amino acid identity) but differ strongly in neuropathogenesis. The immediate-early gene for BICP0 is less conserved (70% amino acid identity) and may contribute to a dissimilar phenotype. A peculiar difference is a guanosine hexamer in the BICP0-1 gene which aligns with only five guanosines in the BICP0-5 gene and therefore results in a frameshift in the latter open reading frame. Thus, the C-terminal amino acid sequence (residues 643–676 of BICP0-1 vs. 655–720 of BICP0-5) is completely different. We introduced the BICP0-5 frameshift into the BoHV-1 genome cloned as a bacterial artificial chromosome (BoHV-1 BAC) using the Red recombination system with galK selection and counterselection. Transfection of MDBK cells with the resulting BAC produced recombinant virus that replicated like wild type BoHV-1 in vitro. Attempts to exchange the entire BICP0-1 gene by the BoHV-5 homolog using the same approach failed repeatedly. Therefore, we cotransfected purified BICP0⁻/galK⁺-BoHV-1 BAC DNA with a recombination plasmid coding for BICP0-5 with or without a HA tag into MDBK cells. BoHV-1 recombinants expressing the respective proteins were characterized. In vitro, all recombinants grew to similar titers as the parental viruses, which demonstrates that BICP0-5 compensates for the growth defect of BICP0⁻/galK⁺-BoHV-1 and functionally complements BICP0-1 of BoHV-1. We conclude that BICP0 may be suitable to positively select BoHV-1 recombinants with deletions or insertions of additional genes of interest.     

Differentiation of vaccine and field strains of bovine herpesvirus type 1 by restriction endonuclease analysis

Bovine herpesvirus type 1 (BHV-1) DNA molecules obtained from a limited number of infected cells were cleaved with a variety of restriction endonucleases. By use of selected DNA fragments in Bgl 1, Pst 1, Pvu 1 and Pvu 11 restriction patterns, one reference, two vaccine and three wild-type strains of BHV-1 were distinguished from one another. The simplified DNA fingerprinting method described here should be most useful, not only to control the genetic stability of BHV-1 vaccines during production, but also to differentiate the vaccine strains from other isolates in clinical cases.     

Hemagglutination inhibition and virus neutralizing response of rabbits inoculated with bovine herpesvirus 1 subunit vaccine

The immunogenicity of bovine herpesvirus type 1 (BHV-1) hemagglutinin has been investigated. Both live and nonionic detergent solubilized vaccines were prepared and 5000 hemagglutinating units (HAU) were injected subcutaneously into rabbits. Both types of vaccine induced a good antibody response but live virus was four times more efficient in inducing hemagglutination inhibiting and neutralizing antibodies than either Triton X-100- or octylglucoside-solubilized subunit vaccine. Blotting analysis revealed that five proteins, of 105,000, 90,000, 74,000, 64,000 and 54,000 mol. wt, were recognized by the serum of vaccinated animals. Triton X-100-solubilized vaccine did not induce antibodies against the 105,000 and 64,000 mol. wt proteins, indicating the important role of VP 90,000 and VP 74,000 in hemagglutination and neutralization. The order in which antibodies to the different viral proteins were induced was VP 90,000, (VP 105,000, VP 64,000, VP 54,000) and VP 74,000. Our data indicate that VP 90,000 is the hemagglutinin. Using convalescent serum from intranasally infected animals, we could identify nine structural proteins for BHV-1; VP 105,000, VP 90,000, VP 74,000, VP 64,000, VP 54,000, VP 50,000, VP 47,000, VP 40,000 and VP 31,000.     

Development and trial of a bovine herpesvirus 1 - thymidine kinase deletion virus as a vaccine

SUMMARY An Australian bovine herpesvirus 1 (BHV1) isolate with a defined (427 base pair) deletion in the protein coding region of the thymidine kinase gene was obtained by standard marker rescue procedures. After selection in the presence of the nucleotide analogue 5iodo-deoxy-uridine the virus was analysed by hybridisation with three differential oligonucleotide probes, restriction endonuclease profile studies and DNA sequence analysis. The virus elicited an immune response in recipient animals after either intramuscular or intravenous administration and produced no significant deleterious side-effects when administered at a dose sufficient to stimulate the host immune response. The safety and immunogenicity of the recombinant BHV1 virus 39B1 were similar to those reported for other registered BHV1 vaccines and the virus would appear to be suitable for the production of a vaccine seed lot and more exhaustive field trials as a prelude to commercial vaccine production and registration.     

Efficacy of a live bovine herpesvirus type 1 marker vaccine under field conditions in three countries

The performance of a live marker vaccine for bovine herpesvirus type 1 (bhv-1) was studied in the field in three European Union countries with different farming conditions. The progress in the eradication of the virus was followed in a large herd in Germany and one in Italy, and a major serological survey involving 147 farms was conducted in Hungary. Commercial batches of the same vaccine were used in all three studies. The herds were vaccinated according to agreed protocols and the animals' bhv-1 antibody status was determined at local institutes by using commercial glycoprotein B (gB)- and glycoprotein E (gE)-elisas. In all three studies, the seroprevalence of bhv-1 gE decreased progressively. Given the starting conditions and the long duration of the studies, reactivation events and virus circulation would have been more likely to have occurred if the herds had not been vaccinated.     

Genetic immunisation of cattle against bovine herpesvirus 1: glycoprotein gD confers higher protection than glycoprotein gC or tegument protein VP8

Bovine herpesvirus 1 (BoHV-1) has frequently been used as a model for testing parameters affecting DNA immunisation in large animals like cattle. However, the selection of target antigens has been poorly studied, and most of the experiments have been conducted in mice. In the present study, we demonstrated in cattle that a DNA vaccine encoding BoHV-1 glycoprotein gD induces higher neutralising antibody titres than vaccines encoding BoHV-1 gC. Additionally, we show that a DNA vaccine encoding a secreted form of gD induces a higher immune response than a vaccine encoding full-length gD. However, the enhanced immunogenicity associated with the secretion of gD could not be extended to the glycoprotein gC. The current study also describes for the first time the development and the evaluation of a DNA vaccine encoding the major tegument protein VP8. This construct, which is the first BoHV-1 plasmid vaccine candidate that is not directed against a surface glycoprotein, induced a high BoHV-1 specific cellular immunity but no humoral immune response. The calves vaccinated with the constructs encoding full-length and truncated gD showed a non-significant tenfold reduction of virus excretion after challenge. Those calves also excreted virus for significantly (p < 0.05) shorter periods (1.5 days) than the non-vaccinated controls. The other constructs encoding gC and VP8 antigens induced no virological protection as compared to controls. Altogether the DNA vaccines induced weaker immunity and protection than conventional marker vaccines tested previously, confirming the difficulty to develop efficient DNA vaccines in large species.     

Patterns of bovine T cell-mediated immune responses to bovine herpesvirus 1

Lymphocyte proliferation was used to evaluate T cell-mediated immune responses to different isolates of Bovine Herpesvirus 1 (BHV-1). Groups of high, moderate, and low responses were observed when lymphocytes from three breeds of dairy cattle were stimulated with each of the BHV-1 isolates. Proliferation of cells in the low responding group could be augmented by exogenous IL-2. The mechanism of unresponsiveness by cells from one individual whose response was not altered by IL-2 supplementation was further investigated. The patterns of response by limiting dilution frequency analysis eliminated the possibility that this individual lacked responsive cells but suggested the presence of regulatory cell interactions which resulted in the observed low proliferative response. These results show that animals exposed to the same environment can vary greatly in their ability to respond to BHV-1. At least two mechanisms may be responsible for low proliferative responses in vitro: inadequate levels of IL-2 and the presence of suppressor cells.     

Development of a polymerase chain reaction and restriction typing assay for the diagnosis of bovine herpesvirus 1, bovine herpesvirus 2, and bovine herpesvirus 4 infections.

A multiplex polymerase chain reaction (PCR) method coupled with a restriction analysis of PCR products (PCR with restriction fragment length polymorphism) was developed for the simultaneous detection of bovine herpesvirus 1, bovine herpesvirus 2, and bovine herpesvirus 4 infections. The specificity, sensitivity, and practical diagnostic applicability of this method were evaluated. This assay may be also adapted to the diagnosis of suid herpesvirus 1 and equine herpesviruses 1 and 3 and could become a powerful diagnostic tool.     

Analysis of bovine trigeminal ganglia following infection with bovine herpesvirus 1

Following primary infection of the eye, oral cavity, and/or nasal cavity, bovine herpesvirus 1 (BHV-1) establishes latency in trigeminal ganglionic (TG) neurons. Virus reactivation and spread to other susceptible animals occur after natural or corticosteroid-induced stress. Infection of calves with BHV-1 leads to infiltration of lymphocytes in TG and expression of IFN-gamma (interferon-gamma), even in latently infected calves. During latency, virus antigen and nucleic acid positive non-neural cells were occasionally detected in TG suggesting there is a low level of spontaneous reactivation. Since we could not detect virus in ocular or nasal swabs, these rare cells do not support high levels of productive infection and virus release or they do not support virus production at all. Dexamethasone (DEX) was used to initiate reactivation in latently infected calves. Foci of mononuclear or satellite cells undergoing apoptosis were detected 6h after DEX treatment, as judged by the appearance of TUNEL+ cells (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling). BHV-1 antigen expression was initially detected in lymphocytes and other non-neural cells in latently infected calves following DEX treatment. At 24h after DEX treatment, viral antigen expression and nucleic acid were readily detected in neurons. Our data suggest that persistent lymphocyte infiltration and cytokine expression occur during latency because a low number of cells in TG express BHV-1 proteins. Induction of apoptosis and changes in cytokine expression following DEX treatment correlates with reactivation from latency. We hypothesize that inflammatory infiltration of lymphoid cells in TG plays a role in regulating latency.