Bovine Herpesvirus 1 Infection Research Articles

Immunoglobulin M (IgM) indirect enzyme-linked immunosorbent assay and the involvement of IgM-rheumatoid factor in the serodiagnosis of BHV-1 infection

An IgM enzyle-linked immunosorbent assay (IgM-ELISA) for the detection of antibodies to bovine herpesvirus-1 (BHV-1) was developed. Its applicability was examined by serological studies in two calves experimentally infected with virulent BHV-1 over a period of 60 days. IgM antibodies were detected by ELISA on day 6 after infection, and there was an increase in IgM antibodies on day 9. Serum neutralizing (SN) antibodies were detected only on day 13, confirming the higher sensitivity of the ELISAs. A similar study of four calves treated with a commercial inactivated virus vaccine indicated no detectable IgM-ELISA response, and late SN and IgG-ELISA reactivity. Thus IgM-ELISA appears to be of value in assessing recent infection, whereas IgG-ELISA and SN cannot distinguish between infection and vaccination. The possible limitations imposed on the specific IgM-ELISA by the presence of IgM rheumatoid factor (IgM-RF) in bovine serum were examined. IgM-RF levels were determined in bovines of various ages. Elevated values of IgM-RF were found in the sera of older animals; their occurrence may lead to false IgM-positive diagnosis (16%) o BHV-1 infection. This was examined in 113 serum specimens from suspected cases of BHV-1 infection and in 32 bulls at an insemination center. Pretreatment of serum samples with an antibovine IgG serum eliminated false positivity of the IgM-ELISA. It is concluded that IgM-ELISA should be of particular value in the diagnosis of recent infection with BHV-1, mainly in calves.

Lymphocyte proliferative responses to separated bovine herpesvirus 1 proteins in immune cattle

The immune response to bovine herpesvirus 1 (BHV-1) infection can protect cattle from subsequent challenge with the virus. This protection involves a variety of defensive strategies, and the activation of most of these defenses requires the recognition of viral proteins by the cellular immune system. To identify some of the BHV-1 proteins recognized by T lymphocytes, we measured in vitro proliferative responses to individual proteins. Viral proteins were separated by gel electrophoresis followed by Western immunoblotting, and immunoblots were evaluated for serological reactions. Unstained blotted fractions were processed into antigen-bearing particles for analysis in blastogenesis assays. Purified BHV-1 proteins obtained by immunoadsorbent chromatography were processed and included for comparison in both enzyme-linked immunosorbent and proliferation assays. The tegument protein VP8 and the glycoprotein gIV appeared to be the antigens which most consistently stimulated the proliferation of lymphocytes from BHV-1-immunized animals. Positive blastogenic responses were also detected to gI, gIII, and to one or more uncharacterized, low-molecular-weight proteins in some of the cattle tested. These results indicate that T-lymphocyte proliferative responses to BHV-1 proteins are detectable in immune cattle and may be important in protection from BHV-1 infection.

Synthesis, cellular location, and immunogenicity of bovine herpesvirus 1 glycoproteins gI and gIII expressed by recombinant vaccinia virus

Two of the major glycoproteins of bovine herpesvirus 1 (BHV-1) are gI, a polypeptide complex with apparent molecular weights of 130,000, 74,000, and 55,000, and gIII (a 91,000-molecular-weight [91K] glycoprotein), which also exists as a 180K dimer. Vaccinia virus (VAC) recombinants were constructed which carry full-length gI (VAC-I) or gIII (VAC-III) genes. The genes for gI and gIII were each placed under the control of the early VAC 7.5K gene promoter and inserted within the VAC gene for thymidine kinase. The recombinant viruses VAC-I and VAC-III retained infectivity and expressed both precursor and mature forms of glycoproteins gI and gIII. The polypeptide backbones, partially glycosylated precursors, and mature gI and gIII glycoproteins were indistinguishable from those produced in BHV-1-infected cells. Consequently, they were apparently cleaved, glycosylated, and transported in a manner similar to that seen during authentic BHV-1 infection, although the processing efficiencies of both gI and gIII were generally higher in recombinant-infected cells than in BHV-1-infected cells. Immunofluorescence studies further demonstrated that the mature gI and gIII glycoproteins were transported to and expressed on the surface of cells infected with the respective recombinants. Immunization of cattle with recombinant viruses VAC-I and VAC-III resulted in the induction of neutralizing antibodies to BHV-1, which were reactive with authentic gI and gIII. These data demonstrate the immunogenicity of VAC-expressed gI and gIII and indicate the potential of these recombinant glycoproteins as a vaccine against BHV-1.

Inhibition of T-lymphocyte mitogenic responses and effects on cell functions by bovine herpesvirus 1

The mitogenic response of bovine peripheral blood mononuclear cells stimulated by concanavalin A (ConA) was suppressed by infectious bovine herpesvirus 1 (BHV-1). Proliferation in response to interleukin-2 (IL-2) by IL-2-dependent lymphocyte cultures was also inhibited by BHV-1. Although inhibition of mitogenesis approached 100%, less than 1 cell in 1,000 was productively infected by BHV-1 in ConA-stimulated cultures. Neither conditioned medium from mitogen-stimulated peripheral blood mononuclear cell cultures nor human recombinant IL-2 reversed suppression by the virus. Infection by BHV-1 did not influence the expression of IL-2 or IL-2 receptor mRNA in ConA-stimulated cultures, nor did it affect the cytolytic capabilities of lymphocytes. The data suggest that the inhibition of T-lymphocyte proliferation is the result of a nonproductive BHV-1 infection.

Detection of Bovine Herpesvirus-1-Specific IgM Using a Capture Enzyme Immune Assay with Isotype-Specific Monoclonal Antibodies

The detection of virus-specific immunoglobulin M (IgM) antibodies in acute-phase serum samples offers the possibility of making an accurate and rapid serologic diagnosis. We have developed a solid-phase capture assay that uses murine monoclonal antibodies specific for bovine IgM to separate the whole IgM fraction of a bovine serum sample. The IgM specific for bovine herpesvirus-1 (BHV-1) is then detected by the addition of viral antigen, which in turn is detected by BHV-1-specific monoclonal antibodies conjugated to horseradish peroxidase. A BHV-1 IgM antibody response was detected during the early postinfection period (7-40 days PI). Bovine herpesvirus-1 IgM antibody was not detected in sera taken from 3 animals following dexamethasone-induced viral reactivation. This method compares favorably with viral isolation, antigen detection in the clinical samples, and paired serology in the diagnosis of BHV-1 infection at a herd level.

Effects of immunization with bovine herpesvirus-1 glycoproteins on bovine herpesvirus-1-induced alteration of bovine neutrophil chemotactic and anti-Pasteurella haemolytica activities.

It has been reported previously that active bovine herpesvirus-1 (BHV-1) infection greatly enhances the susceptibility of cattle to secondary bacterial pneumonia involving Pasteurella haemolytica. The present study examines the possibility that immunization of BHV-1 naive calves with purified BHV-1 glycoproteins would protect them against changes in neutrophil function that might compromise their ability to eliminate P. haemolytica during an active BHV-1 infection. The results show that circulating neutrophil chemotactic activity was generally reduced at 7-8 days after BHV-1 challenge; immunization with a 77 kilodalton BHV-1 glycoprotein (gIV) prevented impairment of neutrophil chemotaxis. BHV-1 infection did not markedly affect the ability of neutrophils to ingest and kill P. haemolytica in vitro. Immunization and challenge with BHV-1 had little effect on the chemiluminescence response of bovine neutrophils to opsonized P. haemolytica in vitro, although in one experiment a marked increase in baseline neutrophil chemiluminescence was observed which may be relevant to understanding the pathogenesis of pulmonary damage that occurs in BHV-1 infected calves.

Bovine herpesvirus 1: rapid diagnosis of infection by direct filter hybridization.

Direct filter hybridization (DFH) was applied as a simple method of nucleic acid hybridization to diagnose bovine herpesvirus 1 (BHV-1) infection without previous purification of nucleic acids from the specimens. The DNA of BHV-1 was cleaved with the restriction endonuclease Pst I and randomly cloned into pKH47 plasmids. The clones were labelled with 32P or biotin and selected on uninfected and infected cells for the highest specific activity to detect BHV-1 infection. Two clones, which detected about 10 infected cells, were selected for the diagnosis of BHV-1 in cattle. On specimens collected during experimental and natural disease, the DFH showed to be in concordance with the standard method of virus isolation. This simple hybridization technique proved to be a sensitive and rapid alternative to virus isolation. Specific diagnosis of BHV-1 infection can be made even in simply equipped laboratories within 10 h.

T lymphocyte population dynamics and function following a primary bovine herpesvirus type-1 infection.

Following a primary bovine herpesvirus-1 (BHV-1) infection the concanavalin A (Con A) induced proliferative responses of peripheral blood T lymphocytes were suppressed. This suppression occurred in the absence of detectible serum suppressor factors, suppressor cell activity or decreased accessory cell function. However, regression analysis demonstrated a significant correlation between the percentage of T lymphocytes present within the peripheral blood mononuclear cell (PBMC) population and the amplitude of Con-A-induced lymphocyte proliferative responses (LPR). Direct evidence that a numerical deficit of responder T lymphocytes was limiting LPR was obtained by using an immunomagnetic microsphere (IMM) negative enrichment protocol to produce a PBMC population with a constant percentage (75 +/- 6%) of T lymphocytes. The Con-A-induced LPR of these enriched T lymphocytes remained constant following BHV-1 infection. Flow cytometric (FC) analysis of PBMC indicated that the decreased percentage of circulating T lymphocytes, associated with BHV-1 infection, was caused primarily by a selective depletion of the BoT8 subset. These FC data were consistent with the indirect evidence of increased TH activity, as indicated by elevated Con A-induced IL-2 production. Thus, 2 to 5 days following viral infection, the circulating T lymphocytes were activated as shown by elevated IL-2 production, increased recombinant bovine IL-2 (rBo

Bovine peripheral blood leukocyte subpopulation dynamics following a primary bovine herpesvirus-1 infection.

Population dynamics of bovine peripheral blood leukocyte subpopulations were quantitated following a primary bovine herpesvirus-1 (BHV-1) infection. Percoll isolated peripheral blood mononuclear cell (PBMC) subpopulations were analyzed using flow cytometry (FC) and cytochemical stains. Between days two to eight post-infection (PI) there was a significant decrease in the percentage of T-cells and nonT/nonB cells which was accompanied by an increased percentage of B-cells and monocytes. These percentages were extrapolated to the number of Percoll isolated PBMC during this period. A decrease in the T-cell population was the primary cause of the observed lymphopenia and a relative increase in the percentage of B-cells. The increased percentage of monocytes was caused by an increased number of circulating monocytes. These monocytes were characterized by an increase in Fc receptor expression, a decrease in plastic and Sephadex-G10 adherence and no apparent change in the level of class II MHC antigen (Ia) expression. Serum cortisol was significantly elevated on day 2 PI and may have been responsible for both the reduction in circulating T-cells and a decrease in the in vitro viability of peripheral blood lymphocytes. The percentage of Ia positive PBMC was increased significantly on day 4 PI. However, on days 4 and 6 PI the summated percentages of monocytes and B-cells (total Ia expressing population) exceeded significantly the actual percentage of Ia positive cells. This apparent suppression of Ia expression did not coincide with the elevated serum prostaglandin E2 concentrations on days 8 and 10 PI.

Isolation of a Herpesvirus Serologically Related to Bovine Herpesvirus 1 from a Reindeer (Rangifer Tarandus)

Serological evidence of exposure of reindeer (Rangifer tarandus) to a virus related to bovine herpesvirus 1 (BHV1) (Synonym: Infectious bovine rhinotracheitis (IBR) virus) has been reported in Canada (El Azhary 1979) and the USA (Dieterich 1981). A serological survey conducted in Finnish Lapland also detected neutralising antibodies to BHV1 in reindeer sera; 23 % of 300 reindeer had detectable antibodies, whereas none of 300 cattle sera from the same region contained antibodies to BHV1 (Ek-Kommonen et al. 1982). There is currently no evidence of BHV1 infection of cattle in Finland, so the isolation and characterisation of the reindeer herpesvirus was of considerable interest. This short communication describes the isolation and preliminary characterisation of a herpesvirus from a reindeer following the administration of dexamethasone.

In Vitroand Systemic Effects of Recombinant Bovine Interferons on Natural Cell-Mediated Cytotoxicity in Healthy and Bovine Herpesvirus-1-Infected Cattle

There is now evidence to suggest that the beneficial effect of treatment of calves with recombinant bovine interferons (IFNs) on the outcome of an infection with bovine herpes-virus-1 (BHV-1) may in part be due to effects other than its direct antiviral activity. This evidence prompted us to study the effect of IFNs on natural cell-mediated cytotoxicity (NC) as a possible mechanism in curtailing clinical disease. In vitro treatment of blood leukocytes not only augmented cytotoxicity to NC-susceptible target cells, but also to cell types usually resistant to killing. In vivo treatment of healthy cattle with IFN-gamma caused a transient rise in NC activity, which was both dose and route dependent. Treatment of calves with IFN-alpha 1 or IFN-gamma prior to BHV-1 infection prevented or diminished the suppression of NC activity usually seen following virus infection. The effect could neither be correlated with serum IFN titers nor with conventional hematologic parameters. A possible mechanism for the IFN effect involving the ontogeny of bovine NC-effector cells and the biological consequences for the local antiviral defense in the lung are discussed.

Experimental infection of rabbits with bovine herpesvirus-4: Acute and persistent infection

A strain of bovine herpesvirus-4 (BHV-4) isolated from bovine cases of mammary pustular dermatitis was used for experimental infection of rabbits. The strain is serologically indistinguishable from the group prototype Movar 33/63 and from the American isolate DN599. Groups of rabbits were inoculated by various routes. Intravaginal and conjunctival inoculations resulted in vulvovaginitis and conjunctivitis, respectively and in shelding of virus. The rabbits seroconverted for the virus, with high titers of antibodies (indirect fluorescent antibody test) that persisted throughout the experiment. Treatment with dexamethasone, beyond the acute infection, did not produce recrudescence of disease or shedding of the virus. Rabbits were killed at various times, from 3 to 6 months post-infection, and the virus was recovered from explant cultures of spleen and by cocultivation of spleen cells with bovine lung cells. These results demonstrate the usefulness of the rabbit as a model for studying the pathogenesis of BHV-4 infection in cattle.

Pathogenesis of Bovine herpesvirus-1 (BHV-1) infections: Interactions of the virus with peripheral bovine blood cellular components

Bovine herpesvirus-1 (BHV-1) is believed to reach the placenta via the hematogenous route as the virus has been recovered from the blood buffy coat cells of experimentally infected cattle. However, the manner in which the virus relates to these and other blood cells while in transit in the blood is not known. The nature of this relationship was investigated in the present study. An in vitro correlate of the in vivo cell-virus interactions was simulated by isolating peripheral blood cells on a Ficoll-Hypaque gradient and testing their capacity to adsorb and act as host cells for BHV-1 replication. It was shown that all leukocytes, but not red cells, can adsorb virus readily. Of the leukocytic fraction, monocytes and lymphocytes adsorb the virus most effectively. Monocytes supported viral replication while lymphocytes could do so only after stimulation with phytohemagglutinin. It is concluded that in vivo replicating BHV-1 is transported in monocytes, and is adsorbed to all leukocytes from which it finally reaches the placenta, leading to abortion.

Persistent infection with bovine herpesvirus-1 (infectious bovine rhinotracheitis virus) in cultured hamster cells

Bovine herpesvirus-1 infection in hamster embryo cells was found to be dependent upon input multiplicity; productive infection was achieved at input multiplicities greater than one, while persistent infection was established when input multiplicities were about 0.5. This persistence was characterized by a noncyclic, minimal degree of cytopathic effect with a low level of released virus. Maintenance of the persistently infected cultures did not require external supportive measures. Subcultivation of the persistently infected cultures led to virus replication followed by CPE and then cell regrowth. With 3 to 4 weeks after subcultivation a persistent infection was re-established. The possible mechanism for the bovine herpesvirus persistence in hamster cells is discussed.