Epitope specificity of the bovine antibody response to the gIII glycoprotein of bovine herpesvirus type 1.

A panel of monoclonal antibodies (MAbs) to BHV-1, specific for gI and gIII glycoproteins and for a nonglycosylated core protein p100, was used to examine the epitope specificity of the immune response to the virus in naturally exposed and experimentally infected cattle. Sera from experimentally infected calves were analyzed in a competition ELISA (C-ELISA). Antibody to an epitope on gIII appeared as early as 4 days post infection, although virus-neutralizing antibody did not appear until day 8 or later. Antibody production peaked at 13 to 18 days post infection but the level of antibody to each gIII epitope varied. Competition by sera from cattle naturally exposed to BHV-1 was analyzed as a function of the virus neutralization (VN) titer of these sera. Competition with most of the MAbs correlated linearly with neutralization titer. However, competition with 2 of the MAbs, one specific for gIII and one specific for gI, was maximal even at the lowest neutralization titer, and competition with another MAb, specific for a non-glycosylated core protein, p100, was negative. Selected sera from the naturally exposed group were also examined by radioimmunprecipitation, and were shown to react predominantly with gI, gIII and gIV glycoproteins and in a few shown to react predominantly with gI, gIII and gIV glycoproteins and in a few MAbs were determined, and it was found that neutralization was enhanced by certain combinations. A mutually exclusive relationship between neutralization enhancement and competition for binding by MAbs (as determined by reciprocal C-ELISA) indicated an epitope-specific, rather than antibody-specific, mechanism for neutralization enhancement.

Evaluation of an antigen-capture ELISA for the detection of bovine herpesvirus type 1 shedding from feedlot cattle.

A total of 457 nasal swab specimens from cases of respiratory disease in 2 feed lots were evaluated for the detection of bovine herpesvirus Type 1 (BHV-1) by ELISA. Thirty-three were found to be positive for BHV-1 by the recovery of infectious virus and 21 of these were positive by ELISA, yielding a sensitivity of 64%. Fifteen other virus isolations were made and included bovine viral diarrhea viruses, rhinoviruses and parainfluenza Type 3 viruses; none of these cases were positive with the BHV-1 ELISA. Specificity of the ELISA was 100%. Eighty percent of the specimens with BHV-1 titers greater than 10(5) TCID50 were detected by ELISA; the median amount of virus in positive specimens that were detected by ELISA was 7 X 10(5) TCID50 and the median amount of virus in specimens not detected was 1.5 X 10(4) TCID50. BHV-1 infection was most frequently diagnosed in feedlot cattle that had been in the feedlot for 40-80 days. Approximately half of the infected cattle were carrying virus-neutralizing antibodies in their serum.

Use of in situ hybridization with a biotinylated probe for the detection of bovine herpesvirus-1 in aborted fetal tissue.

Forty-five cases of bovine abortion were examined using in situ hybridization (ISH) with a biotinylated DNA probe specific for bovine herpesvirus-1 (BHV-1). Of the 45 cases, 16 were diagnosed as due to BHV-1, 15 were determined to be due to other causes, and 14 were of undetermined etiology. Direct comparisons between ISH and an immunoperoxidase (IP) test specific for BHV-1 were performed on formalin-fixed, paraffin-embedded tissue sections of lung, liver, kidney, spleen, thymus, and placenta; fluorescent antibody tests for BHV-1 and virus isolation were performed on fresh lung and liver. In comparison to these routine BHV-1 detection techniques, ISH had an overall sensitivity of 88.2% and a specificity of 89.3% in detecting BHV-1 in aborted fetuses. Immunoperoxidase was more sensitive than ISH with tissue sections from lung (87.5% vs. 69%), liver (92% vs. 17%), spleen, and placenta; results of the tests on tissue sections from kidney were concordant. Liver sections presented special problems in that nonspecific reactions were frequently observed with hybridization. With thymus sections, the rate of detection was higher by hybridization than by IP, but the specificity of some of these reactions could not be confirmed.

Rapid, sensitive, competitive serologic enzyme-linked immunosorbent assay for detecting serum antibodies to bovine herpesvirus type 1.

An enzyme-linked immunosorbent assay (ELISA) for the detection of cattle antibodies to bovine herpesvirus type 1 was developed on the basis of competition between serum antibody and a virus-neutralizing mouse monoclonal antibody. The assay showed improved sensitivity over the virus neutralization (VN) test and over an enhanced VN test in which incubation of antibody-virus mixtures was carried out for 24 h. With the ELISA, antibodies in sera from experimentally infected cattle were detected earlier after infection and showed more rapid increases in levels. A comparison of the ELISA with the VN tests by using a set of 85 field sera with low levels of antibodies demonstrated that the ELISA was the most sensitive test, detecting 10 positive serum samples that were negative by the VN tests. The ELISA was inexpensive, rapid, and highly reproducible and showed a significant improvement in sensitivity over VN tests.

Epitope-specific antibody responses in market-stressed calves to bovine herpesvirus type 1.

Reciprocal competition ELISA (rcELISA) was conducted to map monoclonal antibodies (mAbs) reactive with gI, gIII and gIV glycoproteins of bovine herpesvirus type 1 (BHV-1) into epitope groups. mAbs to glycoproteins gI and gIV were divided into six epitope groups each, while gIII mAbs had been previously divided into four areas. mAbs were chosen from each epitope group to compete in cELISA wih bovine sera collected during a typical regimen of vaccination and transportation from farm to auction to feedlot. The immunodominant epitopes were identified for each BHV-1 glycoprotein. With glycoprotein gI, three epitopes defined by mAbs 1F10, D9 and 4807 were the most dominant; with glycoprotein gIII epitopes defined by mAbs G2 and 1507, and with glycoprotein gIV epitopes defined by mAbs 1102, 1106, 3C1, 3402 and 3E7 showed the maximum responses. The overall cELISA responses to each glycoprotein among two vaccination groups were also compared and it was shown that cELISA responses were significantly higher for each glycoprotein in calves receiving two vaccinations, one on the farm of origin and one at auction, than in calves receiving only one vaccination at auction.

Experimental infection of neonatal calves with neurovirulent bovine herpesvirus type 1.3.

A type of bovine herpesvirus, BHV-1.3, causes encephalitis in calves, whereas BHV-1.1 causes respiratory disease. Three colostrum-deprived calves and two colostrum-fed calves were inoculated with BHV-1.3 by intranasal aerosolization. Two colostrum-deprived calves were inoculated with BHV-1.1 by intranasal aerosolization. BHV-1.3-inoculated calves demonstrated severe encephalitis with minimal respiratory lesions, and BHV-1.1-inoculated calves demonstrated severe respiratory lesions and no clinical signs of neurologic disease. Calves fed colostrum that contained virus neutralizing antibodies were protected against neurologic disease. Colostrum-fed BHV-1.3-inoculated calves did not develop disease although they did become infected; virus was shed in respiratory secretions for 10-13 days postinoculation, similar to infected colostrum-deprived calves. BHV-1.3 was reactivated from a latent state from one colostrum-fed calf after administration of dexamethasone 60 days postinoculation. Histopathologic examination of the three colostrum-deprived BHV-1.3-inoculated calves revealed severe lesions of encephalitis. One of the two BHV-1.1-inoculated calves had one focal lesion of encephalitis. Virus was isolated from brain tissue of colostrum-deprived BHV-1.3-inoculated calves and from one BHV-1.1-inoculated calf. Immunohistochemical staining for BHV-1 antigen was observed in neurons from the colostrum-deprived BHV-1.3-inoculated calves.

Monoclonal antibodies that distinguish between encephalitogenic bovine herpesvirus type 1.3 and respiratory bovine herpesvirus type 1.1.

Seven mouse hybridoma cell lines producing monoclonal antibodies (MAb) against an encephalitogenic strain of bovine herpesvirus type 1.3 (BHV-1.3) were established. The clones producing MAb were selected to be specific for BHV-1.3 by enzyme-linked immunosorbent assay. Only L1B neutralized virus without complement. With the addition of complement, five of the MAb neutralized BHV-1.3 but not the respiratory strain BHV-1.1. The anti-BHV-1.3-specific MAb Q10B, L6G, and L1B precipitated glycoproteins from BHV-1.3 that were analogous to the gI, GIII, and gIV glycoproteins of BHV-1.1, respectively. The other four MAb precipitated unknown proteins. None of the anti-BHV-1.3 MAb precipitated BHV-1.1 glycoproteins. The majority of the anti-BHV-1.3 MAb did not react with BHV-1.1 by immunoblotting, but O7E (unknown protein pattern by radioimmunoprecipitation) was reactive with five proteins (M(r)s of 33,000, 43,000, 70,000, 141,000, and 190,000) of BHV-1.3 and with a different pattern of proteins of BHV-1.1 (M(r)s of 30,000, 38,000, 83,000 and 144,000). Two of the MAb, L6G and O7E, conjugated with peroxidase were found to be useful for detecting BHV-1.3 antigen by immunochemistry in Formalin-fixed brain tissue from experimentally infected calves.

Conformational alteration of Sindbis virion glycoproteins induced by heat, reducing agents, or low pH.

Sindbis virions undergo a conformational rearrangement after attachment to cells but prior to entry, as detected by exposure of epitopes on virus-cell complexes which are not accessible to their cognate monoclonal antibodies on native virions (D. C. Flynn, W. J. Meyer, and R. E. Johnston, J. Virol. 64:3643-3653, 1990). The rearrangement did not appear to require transit of virions through a low-pH environment, and the altered virions participated in a productive infection. This naturally occurring structural alteration could be mimicked, although not precisely duplicated, by any of the three artificial treatments of purified virions in vitro: brief incubation at 51 degrees C, treatment with 1 to 5 mM dithiothreitol, or incubation of pH 5.8 to 6.0. Infectivity was maintained after all three treatments, suggesting that Sindbis virions are metastable and can exist in at least two infectious conformations. The integrity of external, neutralizing epitopes was maintained on cell-associated virions and in the altered conformations induced by heat and dithiothreitol, whereas these epitopes were unreactive under low-pH conditions that induced an analogous exposure of previously inaccessible epitopes. The pH at which the conformational change was induced and the pH at which virions could mediate cell-cell fusion from without were coordinately shifted when these two parameters were determined for another strain of Sindbis virus. This coordinate shift in pH optima suggests that the conformational change in virion structure observed at the cell surface may be causally related to fusion.

Antigenic differences between the major glycoproteins of bovine herpesvirus type 1.1 and bovine encephalitis herpesvirus type 1.3.

Differences in the antigenic structure of the major glycoproteins, gI, gIII and gIV, of bovine herpesvirus type 1.1 (BHV1.1) and the neurovirulent BHV1.3 were demonstrated with a panel of monoclonal antibodies (MAbs) prepared against the BHV1.1 glycoproteins. Glycoprotein gIII of BHV1.3 was the most dissimilar, reacting with only four of 15 gIII-specific MAbs. Glycoproteins gI and gIV of BHV1.3 reacted with eight of 11 and eight of 12 specific MAbs, respectively. Monospecific bovine antisera to the two viruses supported findings from the MAb analysis in that gI and gIV glycoproteins were cross-recognized, but gIII was not. Virus-neutralizing MAbs reactive to each glycoprotein and which reacted with both viruses also neutralized both viruses. Previously undescribed glycoproteins which were antigenically related to the intact gIII glycoproteins, but of reduced sizes and lacking at least one gIII epitope, were found for both viruses. Tunicamycin inhibition experiments and immunoprecipitation data suggested that these proteins were intracellular degradation products. Comparisons of the peptide footprints of the glycoproteins from the two viruses using protease V8 digestion after immunoprecipitation with cross-reactive MAbs revealed distinctive footprint patterns for the respective glycoproteins.

Immunogenicity and protective efficacy of a gE, gG and US2 gene-deleted bovine herpesvirus-1 (BHV-1) vaccine.

The efficacy and safety of a gene-deleted bovine herpesvirus-1 (BHV-1) vaccine was determined in a bovine herpesvirus challenge trial in calves. Three different doses of the vaccine were administered intramuscularly at 10(5), 10(6) and 10(7) PFU/ml and compared to a commercial vaccine and non vaccinated control calves. Challenge was performed by intranasal aerosolization with the Cooper strain of BHV-1 (3 x 10(4) PFU/ml). The non-vaccinated calves shed significantly (P < 0.05) more virus than all other groups on days 4, 8 and 10 post challenge. By day 14 post challenge, antibody titers for BHV-1 of calves vaccinated with 10(7) PFU/ml were significantly (P < 0.05) higher than the commercial or non-vaccinated calves. Clinical scores of non-vaccinated calves were significantly (P < 0.05) higher than all other groups on days 4-14 post challenge. With both radioimmunoprecipitation and competitive enzyme-linked immunosorbent assays (C-ELISA), calves in the gene-deleted vaccine groups mounted comparable specific responses against gB, gC and gD post vaccination as calves in the commercial vaccine group, but in a dose dependent manner. These data suggest that the gene-deleted BHV-1 vaccine tested may be used as an effective vaccine in controlling BHV-1 infections.