Transmission of bovine herpesvirus 1 within and between herds on an island with a BHV1 control programme.

Transmission of bovine herpesvirus 1 (BHV1) within and between herds was studied on the island of Ameland, The Netherlands. There were 50 herds with 3300 head of cattle on the island. Herds were divided into three groups: (1) only containing seronegative cattle, (2) containing seronegative cattle and vaccinated seropositive cattle, and (3) containing only vaccinated cattle. All 23 herds in groups 1 and 2 were monitored. Three major outbreaks of BHV1 infections were observed due to the introduction of infectious cattle. Another major outbreak was most likely induced by reactivation of latent BHV1 in seropositive cattle. The basic reproduction ratio within these herds was estimated at least 4. Only one of these outbreaks led to three secondary outbreaks in susceptible herds in which all cattle were seronegative. These outbreaks were most likely due to respectively, direct animal contact, human transmission, and aerogenic transmission. The basic reproduction ratio between herds in this study was estimated to be 0.6.

Presence of bovine herpesvirus 1 gB-seropositive but gE-seronegative Dutch cattle with no apparent virus exposure.

Two hundred and thirty-seven of 2052 cattle which had not been vaccinated against bovine herpesvirus 1 (BHV-1) were seropositive in a glycoprotein B (gB)-blocking ELISA, but seronegative in a glycoprotein E (gE)-blocking ELISA. In order to detect whether they were latently infected with BHV-1, 10 of them were treated with corticosteroids in an attempt to reactivate putatively latent virus. After successive treatments with dexamethasone and prednisolone, no virus excretion was detected and they showed no increase in antibody titres. In contrast, one gE-seropositive animal re-excreted BHV-1 and had a four-fold increase in antibody titre after the corticosteroid treatments. After slaughter, no BHV-1 DNA could be detected with a sensitive PCR in samples of the trigeminal, cervical and sacral ganglia and spinal cords of the gE-seronegative cattle.

Epitopes on glycoprotein E and on the glycoprotein E/glycoprotein I complex of bovine herpesvirus 1 are expressed by all of 222 isolates and 11 vaccine strains.

Glycoprotein E (gE) of bovine herpesvirus 1 (BHV1) forms a complex with glycoprotein I (gI) and plays an important role in cell-to-cell spread mechanisms of the virus, but is not essential for propagation of the virus. To study the antigenic variability of BHV1 glycoprotein E, a set of six well characterised monoclonal antibodies (MAbs) was established using BHV1 gE and gI deletion mutants, eukaryotically expressed gE and gI and pepscan analysis. Two of these MAbs reacted with a linear gE epitope (MAbs 3 and 52), two reacted with a more conformation dependent gE epitope (MAbs 61 and 81) and two reacted with epitopes formed by a complex formed between gE and glycoprotein I (MAbs 67 and 75). With these six MAbs the gE expression of 222 BHV1 isolates and 11 BHV1 modified-live vaccine strains was studied in vitro, using an immunoperoxidase monolayer assay. All 222 BHV1 isolates and 11 vaccine strains were found to react with MAbs 61, 81 and 75. Three of the 222 isolates failed to react with MAb 67 and two of the vaccines reacted very weakly with MAbs 3 and 52. Analysis of the gE genes of these five aberrant isolates and the gE glycoproteins they expressed, did not show obvious size differences compared to wild-type BHV1. We conclude that the tested gE epitopes are highly conserved, including the epitopes formed by the gI/gE complex.

Epitopes on glycoprotein C of bovine herpesvirus-1 (BHV-1) that allow differentiation between BHV-1.1 and BHV-1.2 strains.

In cattle, bovine herpesvirus-1 (BHV-1) can cause a mild genital disease known as infectious pustular vulvovaginitis (IPV) and a more severe respiratory disease known as infectious bovine rhinotracheitis (IBR). On the basis of epidemiological data, it has been proposed that these diseases are caused by strains with different genotypes (IBR by BHV-1.1 and IPV by BHV-1.2 strains). By using a panel of 237 BHV-1 isolates, a monoclonal antibody (MAb 71) was found that failed to react with all 54 putative IPV strains in the panel, and another MAb (77) was found that did not react with 16 of these 54 IPV strains. Because MAbs 71 and 77 also failed to react with a BHV-1.1 glycoprotein C (gC)-deletion mutant, it was hypothesized that both MAbs recognize BHV-1.1 gC. By marker-rescue experiments and by expressing fragments of the BHV-1.1 gC gene in recombinant baculoviruses, it was shown that both MAbs indeed recognize BHV-1.1 gC. MAb 71 recognizes the N-terminal half and MAb 77 recognizes the C-terminal half of BHV-1.1 gC. In a PEPSCAN analysis with 12-mer oligopeptides, MAb 71 reacted with overlapping peptides containing gC amino acid residues 75-80 and MAb 77 did not react in this analysis. The differences in gC found in this study may contribute to the biological differences between BHV-1.1 and BHV-1.2.

Strains of bovine herpesvirus 1 that do not express an epitope on glycoprotein E in cell culture still induce antibodies that can be detected in a gE-blocking ELISA.

Two bovine herpesvirus 1 (BHV1) field strains that do not express an epitope on glycoprotein E (gE) in cell culture were inoculated into calves to examine whether their sera became positive in a gE-blocking ELISA that detects antibodies against gE. This gE-blocking ELISA uses one monoclonal antibody that is directed against the above mentioned epitope. All calves, except one, infected with these gE-epitope negative BHV1 strains, became positive in this gE-blocking ELISA, about two weeks later than in another gE-ELISA and a gB-ELISA. However, cattle infected with BHVI strains that do express this particular gE-epitope showed a similar type of antibody responses. These findings demonstrate that BHV1 strains that do not express a particular gE-epitope in cell culture, still can induce antibodies that are detected in a blocking ELISA that measures antibodies against that epitope.

Reactivation of latent bovine herpesvirus 1 in cattle seronegative to glycoproteins gB and gE.

Six heifers were vaccinated intranasally with the live bovine herpesvirus 1 (BHV1) temperature-sensitive (ts) vaccine strain RBL106 within 3 weeks of birth. These calves most likely still had maternal antibodies against BHV1. Thereafter, these heifers were vaccinated several times with an experimental BHV1 glycoprotein-D (gD) subunit vaccine. At the age of 3 years these 6 heifers were seronegative in the BHV1 gB and gE blocking ELISAs, but had neutralizing antibodies against BHV1, probably induced by the vaccinations with the gD subunit vaccine. Five of these 6 heifers excreted BHV1 after treatment with dexamethasone. Restriction enzyme analysis of the genome of the excreted viruses revealed that all 5 isolates had a BHV1.1 genotype and that isolates of 3 heifers were not obviously different from the ts-vaccine strain. The restriction enzyme fragment pattern of the isolate of 1 heifer was clearly different from the pattern of the ts-vaccine strain. It is concluded that cattle can be seronegative against BHV1 gB and gE but can still carry BHV1 in a latent form. This finding strongly suggests that there are completely BHV1 seronegative cattle that are latently infected with BHV1. The impact of this finding on BHV1 eradication programmes is discussed.

An inactivated gE-negative marker vaccine and an experimental gD-subunit vaccine reduce the incidence of bovine herpesvirus 1 infections in the field.

An inactivated glycoprotein E-negative vaccine and an experimental glycoprotein D-subunit vaccine against bovine herpesvirus 1 (V1) were examined for their effectiveness in a randomized, double-bline, placebo-controlled field trial comprising 130 dairy farms. The use of these marker vaccines enabled us to monitor the incidence of infections in vaccinated populations. The aims of this trial were to evaluate whether these vaccines: (1) reduce the proportion of outbreaks in dairy herds; and (2) reduced virus transmission within dairy herds and to what extent. Vaccination with either of the two vaccines significantly reduced the proportion of herds wherein an outbreak occurred as well as the virus transmission within herds, as compared to placebo-treated herds. The estimated number of secondary cases caused by one infectious animal, expressed as the reproduction ratio R, was for both vaccines significantly > 1. This indicates that when BHV1 is introduced into vaccinated herds, major outbreaks may still occur.

An enzyme-linked immunosorbent assay to detect antibodies against glycoprotein gE of bovine herpesvirus 1 allows differentiation between infected and vaccinated cattle.

A blocking enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against glycoprotein gE (gE) of bovine herpesvirus 1 (BHV1). The assay is based on the use of two monoclonal antibodies directed against different antigenic domains on gE. Sera from uninfected cattle and cattle that had been repeatedly vaccinated with gE-negative marker vaccines scored negative, whereas sera from cattle naturally or experimentally infected with BHV1 field strains scored positive in the gE-ELISA. Antibodies against gE appeared in the serum around 11 days after infection. Cattle that were first vaccinated and then challenged, thus having less virus replication, also became gE-seropositive. The sensitivity and specificity of the gE-ELISA is high, and therefore the gE-ELISA is suitable for differentiating between infected cattle and vaccinated cattle with a gE-negative vaccine.

A simple, specific, and highly sensitive blocking enzyme-linked immunosorbent assay for detection of antibodies to bovine herpesvirus 1.

By using a monoclonal antibody directed against an epitope located on glycoprotein B of bovine herpesvirus 1 (BHV1), a simple, convenient blocking enzyme-linked immunosorbent assay (ELISA) which combines a high sensitivity with a low false-positive rate has been developed. The test can be performed at low variance on undiluted bovine serum samples. The epitope on glycoprotein B appears to be conserved, because it could be detected by immunostaining in all of 160 BHV1 isolates originating from 10 countries. In testing 215 anti-BHV1 antibody-negative and 179 anti-BHV1 antibody-positive serum samples, specificity and sensitivity were 0.96 and 0.99, respectively. This blocking ELISA is superior to a commercially available indirect ELISA and to the 24-h virus neutralization test in detecting low antibody levels in serum. In addition, this blocking ELISA is able to detect specific antibodies in serum as early as 7 days postinfection. To minimize any risk of introducing latent BHV1 carriers among noninfected cattle, this blocking ELISA would be, in our opinion, the test of choice.