Analysis of symptoms associated with bovine herpesvirus 1 vaccination

Between 1 May 1998 and 22 February 1999, it was compulsory for Dutch cattle farmers to take measures against bovine herpesvirus 1 (BHV1). Cattle on farms that were not certified as infectious bovine rhinotracheitis (IBR)-free had to be vaccinated twice a year. During the vaccination programme, both farmers and veterinarians reported side-effects of the vaccine. These reports were collected by the Stichting IBR/BVD Schade (SIS; Foundation for IBR/BVD Damage) in order to draw up a damage report. In 1999 in total 6977 cattle farmers lodged complaints which they considered to be related to the vaccination against BHV1. On these farms, 15,150 herd vaccinations had been performed, 10,269 of which were associated with one or more symptoms. During the compulsory vaccination period, 13% of the herd vaccinations led to symptoms and complaints. In March 1999, a number of vaccine batches were found to be contaminated with bovine virus diarrhoea (BVD) virus. For the purposes of this analysis, a 'known contaminated' herd vaccination was defined as one in which at least one 'known contaminated' batch or lot of vaccine was used. In total, 987 of 1007 herds vaccinated with 'known contaminated' vaccines developed one or more symptoms compatible with acute BVD. There were no commonly seen combinations of symptoms. For this reason, and because the start and end dates were not reported for 55% of the symptoms, it was not possible to detect a symptom pattern. Therefore there were no 'suspect' batches of vaccine which, although not contaminated with BVD virus, gave rise to symptoms. The number of BVD symptoms was determined for those herds with vaccination-related symptoms. There was no difference in the distribution frequency between batch numbers or between 'known contaminated' batches and 'non-suspect' batches. The farmers' definition of chronic wasting was used in this investigation, with the inevitable large differences in definition. The symptom chronic 'wasting' was reported for 3209 of the 10,269 herds with vaccination-related symptoms. On 161 farms (164 herd vaccinations) 'chronic wasting' accounted for more than 20% of the symptoms. As expected, other symptoms were reported in addition to wasting. The symptom 'chronic wasting' was reported more often on forms where a 'known contaminated' vaccine was used. Inactivated vaccine was used for 154 herd vaccinations. In 34 cases, one or more symptoms of acute BVD were reported. The frequency was the same as that for live vaccines. The frequency of reported symptoms tended to be lower with the inactivated vaccine. On the basis of the SIS data, no relationship was found between vaccine batch and reported symptoms. This may be because (i) the classification of a vaccine as 'known contaminated', 'non-suspect', and 'not known' may not have been in keeping with the real status of the vaccine, (ii) farmers may have reported symptoms selectively, and (iii) there is no relationship with vaccination against BHV1.     

Study of bovine herpesvirus type 1 strains with monoclonal antibodies.

Fourteen strains of bovine herpesvirus type 1 (BHV-1, IBRV) representing all three groups of BHV-1 (BHV-1.1, BHV-1.2, BHV-1.3) were studied by ELISA using 106 monoclonal antibodies (Mabs) produced against BHV-1. On the basis of the ELISA, the Mabs could be divided into three groups. The first group (40 Mabs, 38%) reacted with all strains, the second group (43 Mabs, 41%) with the respiratory and genital strains (BHV-1.1 and BHV-1.2) while the third group (23 Mabs, 22%) only with the respiratory strains. Only 5 out of the antibodies neutralized respiratory and genital strains, and none of them neutralized the encephalitogenic strains (1.3). Three Mabs selected from each of the 3 groups, and the above five neutralizing strains were studied by Western blot. Antibodies of groups 1 and 3, and two neutralizing antibodies bound to a 90k protein (gpIII), whereas members of group 2 and 3 neutralizing antibodies reacted with a 74k and a 130k protein (both gpl). The results indicate that reactivity with monoclonal antibodies is as suitable for the classification of BHV-1 strains as is restriction endonuclease (RE) analysis but it cannot distinguish between subgroups within the groups.     

Mammary lesions associated with bovine herpesvirus type 4 in a cow with clinical mastitis

Intranuclear eosinophilic inclusion bodies were seen in the lactiferous duct and sinus epithelium of mammary tissues collected from a cow with clinical mastitis. Transmission electron microscopy revealed herpesvirus particles in these cells. Immunolabeling against anti bovine herpesvirus type 4 (BHV-4) rabbit serum was detected in nuclei that had intranuclear inclusion bodies. In addition, BHV-4 was isolated from the mammary tissue. The viral DNA was detected by nested PCR from the same tissue. This is the first report to describe mammary lesions in association with BHV-4.     

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.     

缺失gE基因的牛Ⅰ型疱疹病毒构建及其体外增殖特性比较

为构建gE基因缺失的牛Ⅰ型疱疹病毒(BHV1),本研究在BHV1全基因组感染性细菌人工染色体克隆pBHV1-ZJ的基础上,利用Red E/T重组技术,分别构建了gE基因缺失、gE和gN双基因缺失的pBHV1突变体,通过转染获得了重组病毒rBHV1-△gE,而双基因缺失突变体pBHV1-△gN-△gE则未能观察到细胞病变。病毒噬斑大小和生长曲线试验表明,在感染细胞内和培养基中,rBHV1-△gE滴度与亲本毒株BHV1无明显差异,而rBHV1-△gN显著降低;重组病毒rBHV1-△gE和前期构建的rBHV1-△gN形成的噬斑均显著小于亲本病毒,其大小分别为亲本病毒的18%和64%。rBHV1-△gE和rBHV1-△gN的构建将有助于阐明gE与gN协同作用机制和为研制BHV1新型基因标记疫苗奠定基础。     

Encephalitis in aborted bovine fetuses associated with Bovine herpesvirus 1 infection.

Brain tissue from 12 aborted bovine fetuses submitted to the Wisconsin Veterinary Diagnostic Laboratory revealed histologic lesions that consisted of glial nodules and variable degrees of mononuclear inflammation, microhemorrhage, neuronal necrosis, and cerebral cortical cavitation. A diagnosis of Bovine herpesvirus 1 (BHV-1) abortion had been made in all of these cases through multiple testing modalities. Brain tissue from 8 of the 12 fetuses was immunohistochemically stained with a monoclonal antibody specific to BHV-1, and, in 5 fetuses, there was positive intralesional staining of neurons, glial cells, and endothelial cells. This preliminary data suggested that herpesviral infection of brain tissue led to the described neurologic lesions. BHV-1 was then amplified from brain tissue in all 12 of the fetuses and was confirmed by partial sequencing of the thymidine kinase and glycoprotein C genes. To the authors' knowledge, neurologic lesions have not previously been described in BHV-1-infected fetuses, nor has BHV-1 previously been identified in bovine fetal brain tissue. The neurologic histopathology attributed to BHV-1 infection in these cases overlaps with the neurologic lesions produced by Neospora caninum, a common etiologic agent of bovine abortion. Therefore, when bovine fetal neurologic lesions are found, both etiologies should be considered and then distinguished by using additional diagnostic tools.     

Effect of genistein on replication of bovine herpesvirus type 1

OBJECTIVE: To study the antiviral activity of genistein, a soya isoflavone, on in vitro replication of bovine herpesvirus type 1 (BHV-1). SAMPLE POPULATION: Madin-Darby bovine kidney (MDBK) cells. PROCEDURE: Effects of genistein on the magnitude and kinetics of inhibition of BHV-1 phosphorylation of glycoprotein E (gE) and in vitro replication of BHV-1 in MDBK cells were evaluated. Antiviral activity of genistein was compared with 2 compounds, estradiol-17beta (EST) and tamoxifen (TAM), that have estrogenic and antiestrogenic activity, respectively. High-performance liquid chromatography (HPLC) was used to determine the concentration of genistein in medium from infected and uninfected MDBK cultures. RESULTS: Genistein reduced BHV-1, but not gE-deleted BHV-1 (BHV-1gEdelta3.1), replication by 90% at 18 hours after inoculation. This inhibition was not sustained through 24 hours after inoculation. The genistein concentration in media from MDBK cells was decreased by 40% during BHV-1 infection, compared with 16% for uninfected cells, at 24 hours after inoculation. Genistein inhibited gE phosphorylation and BHV-1 replication in a dose-dependent manner. Dosing with 25 microM genistein at 0 and 12 hours after inoculation of BHV-1 was optimal for decreasing BHV-1 replication. Estradiol-17beta EST and TAM did not affect BHV-1 replication. CONCLUSIONS AND CLINICAL RELEVANCE: The decrease in genistein concentration was a viral infection-dependent event. Genistein is an inhibitor of BHV-1 replication because of its ability to inhibit tyrosine kinase activity. A possible application may be for the control of BHV-1 infection in cattle by feeding soya products rich in genistein prior to or during periods of stress.     

Serological survey of bovine herpesvirus type 1 infection in China

To understand the nationwide seroprevalence of bovine herpesvirus type 1 (BoHV-1) infection of cows in China, 1344 sera of dairy cows from 29 provinces and 765 sera from 6 herds in Hubei province were collected with stratified random sampling. Another 483 sera from imported cows were included. The serum antibody was tested by BoHV-1 gG ELISA. The results demonstrated that the overall nationwide seroprevalence was 35.8% (481/1344), while the prevalence for individual province ranged from 12.1% to 77.8%. Although each province had positive samples, the prevalence was clustered in areas based on the cow population size. In Hubei Province, the overall seroprevalence was 22.2% (170/765) while the prevalence for individual farms varied greatly from 0.0% to 41.5%. The sera from imported cows had a moderate prevalence of 21.7% (105/483).     

Restriction endonuclease patterns of bovine herpesvirus type 1 isolated from bovine mammary glands

Restriction endonuclease analysis was used, in conjunction with viral neutralization and growth-curve experiments, to compare a bovine herpesvirus type 1 (BHV-1) isolate, originally obtained from bovine mammary gland lesions, with a standard BHV-1 strain, infectious bovine rhinotracheitis virus. Although differences were not detected by viral neutralization or growth-curve experiments, restriction fragment patterns generated by Bam HI, Eco RI, Hind III, and Hpa I, revealed definite differences between the isolate and the prototype strain. Additionally, Eco RI, Hind III, and Hpa I patterns revealed that the mammary gland isolate had DNA-fragment patterns characteristic of infectious pustular vulvovaginitis strain of BHV-1, type 2b. Seemingly, type-2b isolates, similar to types 1 and 2a, may be capable of causing divergent types of infection of variable severity in cattle.     

Uterine infection with bovine herpesvirus type 4 in dairy cows

Diseases of the reproductive tract are a frequent problem in dairy herds. Herpesviruses are uterine pathogens also involved in other clinical diseases; for example, bovine herpesvirus type 4 BoHV‐4 induces abortion, enteritis, metritis, pneumonia and vaginitis, but it can also be detected in healthy cows. The role of BoHV‐4 in the development of clinical endometritis (CE) or subclinical endometritis (SE) has not clearly been described. Therefore, the objective of this study was to describe the prevalence of uterine BoHV‐4 infection and its relationship with clinical, bacteriological and cytological findings in dairy cows 20–30 days after calving. The experiment was performed as a completely randomized block design, with farm (n = 10) as blocking criterion and with cow (n = 397) as the experimental unit. Logistic regression models were used to assess the effect of BoHV‐4 infection on CE, SE and reproductive performance. Proportion of cows infected with BoHV‐4 was 5.8% (n = 23/397). BoHV‐4 was isolated in 11.0% (n = 12/109), 4.8% (n = 4/84) and 3.6% (n = 7/194) of cows diagnosed as CE, SE or healthy, respectively. A logistic model revealed that BoHV‐4 infection showed a tendency to increase the risk for CE (AOR = 2.17; p = .10) but significantly reduced both, the odds for artificial insemination within 80 days post‐partum (dpp) (AOR = 0.37; p = .035) and for pregnancy within 200 dpp (AOR = 0.13; p = .004). Furthermore, BoHV‐4 infection increased the chance for intrauterine infection with Trueperella pyogenes (AOR = 5.55; p < .001) and vice versa (AOR = 5.79, p < .001). In conclusion, BoHV‐4 infection is associated with reduced chances for insemination and pregnancy by 200 dpp and showed a trend to be associated with increased risk for CE. Furthermore, BoHV‐4 and Trueperella pyogenes infections are strongly related.     

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.     

Growth activity of bovid herpesvirus 1 in bovine follicular oocytes with cumulus cells.

Bovine follicular oocytes collected from bovine ovaries were exposed to bovid herpesvirus 1 (BHV-1). After washings, these oocytes were cultured to mature. As a result BHV-1 could not be removed from the oocytes and could replicate in the oocytes with cumulus cells, but not in the oocytes without the cells. Moreover, the specific fluorescence for BHV-1 was detected in the cumulus cells by a indirect immunofluorescent technique. Therefore these findings suggested BHV-1 could be absorbed in the oocytes but the replication of BHV-1 was done in the cumulus cells.     

Detection of bovine herpesvirus type 4 antibodies and bovine lymphotropic herpesvirus in New Zealand dairy cows

AIM: To detect the presence of bovine herpesvirus (BoHV) type 4 in New Zealand dairy cows with clinical metritis.

METHODS: Serum samples taken from 92 dairy cows with clinical metritis, each from a different farm, were tested for the presence of antibodies against BoHV-4 using a commercially available, indirect ELISA. Peripheral blood mononuclear cells (PBMC) were collected from 10 BoHV-4 seropositive cows, and PBMC were examined by a pan-herpesvirus nested PCR to detect herpesvirus. PCR products were sequenced directly and a proportion of the PCR products were cloned and sequenced to identify the virus present.

RESULTS: Antibodies to BoHV-4 were detected in 23/92 (25%) serum samples. The pan-herpesvirus PCR was positive in 8/10 PBMC samples. Cloning and sequencing identified that all of the eight PCR-positive PBMC contained bovine lymphotropic herpesvirus (BLHV); no BoHV-4 DNA was detected.

CONCLUSIONS: This study reports the finding of the presence of apparent antibodies to BoHV-4, and BLHV DNA in New Zealand dairy cows affected by metritis.

CLINICAL RELEVANCE: Bovine herpesvirus type 4 and BLHV are reported to have the potential to cause reproduction failure in cows. This is the first report of apparent BoHV-4 antibodies, and BLHV in New Zealand. The importance and epidemiology of these viruses in cattle in New Zealand requires further investigation.     

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.     

Antiapoptotic activity of bovine herpesvirus type-1 (BHV-1) UL14 protein

Viruses have evolved different strategies to interfere with apoptotic pathways in order to halt cellular responses to infection. One previous study showed that transient transfection of bovine herpesvirus type-1 (BHV-1) UL14 protein is efficient in protecting Madin Darby kidney (MDBK) and human chronic myelogenous leukemia (K562) cells from sorbitol-induced apoptosis. This protein corresponds to a putative protein of BHV-1, which shares aminoacid sequence with a part of the peptide-binding domain conserved in human heat shock protein (HSP70) family. The pBK-CMV-UL14 plasmid transfected MDBK cells treated with sorbitol did not show caspase-3 and caspase-9 activation with respect to non-transfected MDBK cells (UL14 negative). Furthermore, we report that the expression of the full length sequence of BHV-1 UL14 is evident after 7 h of infection of BHV-1 on MDBK cells which were then treated with sorbitol. These results indicate that UL14 gene product has important implications to enhance cell survival in response to apoptotic stimuli.     

Encephalitis induced by bovine herpesvirus 5 and protection by prior vaccination or infection with bovine herpesvirus 1.

Calves were intranasally challenged with bovine herpesvirus 5 (BHV5) and followed for the development of viral infection, clinical encephalitis, histologic lesions in the brain, and viral sequences in the trigeminal ganglia. Calves that were previously vaccinated with bovine herepesvirus 1 (BHV1, n = 4) or previously infected with BHV1 (n = 5) or that had not been exposed to either virus (n = 4) were compared. No calf developed signs of encephalitis, although all calves developed an infection as indicated by nasal secretion of BHV5 and seroconversion to the virus. Histologic lesions of encephalitis consisting of multifocal gliosis and perivascular cuffs of lymphocytes were observed in calves not previously exposed to BHV1. BHV5 sequences were amplified from the trigeminal ganglia of calves previously vaccinated and from calves not previously exposed to BHV1; calves sequentially challenged with BHV1 and later BHV5 had exclusively BHV1 sequences in their trigeminal ganglia. Administration of dexamethasone 28 days after BHV5 challenge did not influence clinical disease or histologic lesions in either previously unexposed calves (n = 2) or previously immunized calves (n = 2), although it did cause recrudescence of BHV5, as detected by nasal virus secretion.     

Persistent cutaneous ulcers associated with feline herpesvirus type 1 infection in a cheetah.

Persistent cutaneous ulcers developed in a female cheetah cub after an episode of rhinotracheitis. When they were 3 weeks old, the cub and a male littermate developed mucopurulent oculonasal discharge consistent with feline herpesvirus type 1 infection (feline viral rhinotracheitis). The male cub was weaned and its lesions resolved. The female cub remained with the dam until the cub was 3 months old, at which time plaque-like lesions developed on the eye margins and muzzle. These plaques regressed over the next month and were replaced with cutaneous ulcers ranging from 1 to 10 mm in diameter. Feline herpesvirus type 1 was isolated from biopsy specimens collected from the ulcers. Cutaneous ulcers are uncommon manifestations of feline herpesvirus infections and have not been reported in other exotic fields. A proposed susceptibility to viral infections related to low genetic diversity has been proposed in cheetahs, and may be involved in the pathogenesis of persistent herpetic ulcers.     

Restriction endonuclease analysis of a porcine isolate of bovine herpesvirus type 1.

Deoxyribonucleic acid (DNA) was extracted from bovine herpesvirus type 1 (BHV-1) isolated from a stillborn porcine fetus, from the Cooper reference strain of BHV-1, and from an Ontario bovine respiratory isolate. Each DNA was digested with the restriction endonucleases HindIII, EcoRI, HpaI and BamHI. Except for very minor differences in the patterns produced after digestion with EcoRI and HindIII, the DNA of the porcine isolate reacted in a similar manner to the bovine viruses, and it was concluded that the porcine virus is genetically similar to bovine isolates of BHV-1.