Variability occurs in the inverted repeat region of genomic DNA from bovine herpesvirus 1 respiratory, genital and bovine herpesvirus 5 encephalitic isolates

Restriction fragment length polymorphisms (RFLPs) were detected within BHV1.1, BHV1.2, and BHV5 genomes using the restriction enzyme PstI. The genomic areas of these changes has not been previously reported. Using Southern blot hybridization with DNA probes representing the entire genome of BHV1.1, areas of genomic variation were located for a respiratory isolate (BHV1.1), four vaccine isolates (BHV1.1), a genital isolate (BHV1.2), and two encephalitic isolates (BHV5). The most frequently observed RFLPs of BHV1.1 and BHV1.2 occurred within the internal repeat region and the left terminus of the unique long region. When two separate isolates of the encephalitic BHV5 were compared, RFLPs were detected in the internal and right terminal repeat regions. These are the regions of each genome from which immediate early genes are transcribed. No genomic variation was observed throughout the unique long and unique short regions for all BHV1 and 5 isolates examined.     

Structural and functional analysis of bovine herpesvirus 1 minor glycoproteins

Topoisomerases are enzymes that catalyse the transient breakage and rejoining of either one (topo I) or two (topo II) DNA strands, to allow one strand to pass through another and prevent unresolvable tangles during processes such as DNA replication. A number of important clinical antitumour agents act through inhibition of topo II enzymes, while some topo I inhibitors appear likely to enter clinical use. Although these chemicals do not covalently interact with DNA, they have strong mutagenic potential, generally causing events at the level of the chromosome rather than that of the gene. Most are recombinogens, may affect gene expression and can also lead to aneuploidy through effects on chromosome segregation. Most topo I and topo II inhibitors primarily cause mutagenic events associated with the replication fork. However, at least in mitotic chromosomes, topo II enzymes are located at the base of chromosome loops, and topo II inhibitors may facilitate subunit exchanges, leading to major deletions and illegitimate recombinational events. There is evidence that programmed cell death provides an alternative pathway to mutagenesis following treatment by either topo I or topo II inhibitors. The final fate of the cell will result from a balance between these two processes.     

Neuropathology of bovine herpesvirus type 5 (BHV-5) meningo-encephalitis in a rabbit seizure model

CI-994 (acetyldinaline) is an orally active anticancer drug currently in Phase 1 clinical trials. To assess its preclinical toxicity, CI-994 was administered orally as suspensions to Wistar rats (10/sex/dose) and in capsules to beagle dogs (3/sex/dose) once daily for two weeks. Doses were 1.5, 5, and 15 mg/kg for rats (9, 30, and 90 mg/m2, respectively), and 0.5, 2, and 5 mg/kg for dogs (10, 40, and 100 mg/m2, respectively). Systemic exposure was dose-proportional based on toxicokinetic analysis in dogs. Severe clinical signs and mortality occurred at the highest dose in both species beginning on Day 10. Neutropenia, lymphocytopenia, thrombocytopenia, lymphoid depletion, bone marrow hypocellularity, and testicular degeneration were observed in both species, primarily at the mid- and high-doses. Despite continued treatment, neutrophil counts in dogs returned to control levels in Week 2. Other microscopic findings in rats included splenic hematopoietic depletion at all doses and epithelial cell necrosis in various tissues at 15 mg/kg. Additional bone marrow changes in dogs involved myeloid and megakaryocyte hyperplasia at 2 mg/kg and abnormal myeloid and megakaryocyte maturation at 2 and 5 mg/kg. Except for the testicular effects in both species, all changes were reversible within a 4-week (rat) or 9-week (dog) recovery period. The results of these studies show that target organ effects of CI-994 principally involve tissues with rapidly dividing cell populations and that bone marrow suppression is the dose-limiting toxicity. CI-994 also seems to interfere with the release and/or maturation of cells in the bone marrow.     

Detection of bovine herpesvirus 1 (BHV-1) genomic sequences in bovine semen inoculated with BHV-1 by polymerase chain reaction

Polymerase chain reaction (PCR) technique was applied to detect BHV-1 in bovine semen inoculated with BHV-1. The technique was found to be 10(6) times more sensitive than a non-isotopic dot-blot hybridization method in detecting viral genomic DNA. Of the three primer pairs used, the one chosen from glycoprotein gC appeared to be most sensitive as it could detect up to 0.01 TCID50 of BHV-1 in the semen. The technique could be useful in screening breeding bulls or samples of frozen semen prior to use in artificial insemination.     

Detection of a novel bovine lymphotropic herpesvirus

Degenerate PCR primers which amplify a conserved region of the DNA polymerase genes of the herpesvirus family were used to provide sequence evidence for a new bovine herpesvirus in bovine B-lymphoma cells and peripheral blood mononuclear cells (PBMC). The sequence of the resultant amplicon was found to be distinct from those of known herpesvirus isolates. Alignment of amino acid sequences demonstrated 70% identity with ovine herpesvirus 2, 69% with alcelaphine herpesvirus 1, 65% with bovine herpesvirus 4, and 42% with bovine herpesvirus 1. Phylogenetic analysis placed this putative virus within the tumorigenic Gammaherpesvirinae subfamily, and it is tentatively identified as bovine lymphotropic herpesvirus. This novel agent was expressed in vitro from infected PBMC, and cell-free supernatants were used to transfer infection to a bovine B-cell line, BL3. Analysis, with specific PCR primers, of DNA from bovine PBMC and lymphoma cells identified infection in blood of 91% of adult animals (n = 101), 63% of lymphomas (n = 32), and 38% of juveniles (n = 13). Of the adults, herpesvirus infection was present in 94% of animals that were seropositive for bovine leukemia virus (BLV) (n = 63) and in 87% of BLV-seronegative animals (n = 38). Of the seropositive group, 17 animals exhibited persistent lymphocytosis, and 100% of these were herpesvirus positive by PCR. A role for bovine lymphotropic herpesvirus as a cofactor in BLV pathogenesis is considered.     

Abortion in heifers inoculated with a thymidine kinase-negative recombinant of bovine herpesvirus 1

The Copper isolate of bovine herpesvirus 1 (BHV-1) was used to produce a thymidine kinase-negative (TK-) recombinant by insertion of a beta-galactosidase (bgal) expression cassette into the TK coding region. The recombinant virus (TK- bgal+) was tested for abortifacient activity in cattle by inoculation of 5 pregnant heifers at 25 to 29 weeks gestation. Five additional heifers were inoculated with the Cooper TK-positive (TK+) virus to serve as controls. After inoculation, both groups of heifers developed similar febrile responses and neutralizing antibody titers. Virus was isolated from blood of all heifers during the first postinoculation (PI) week, and isolation frequencies were similar for both groups. In contrast, whereas virus was isolated from many of the nasal and vaginal swab specimens of heifers inoculated with TK+ virus, only rare virus isolations were made from the heifers given TK- bgal+ virus. All heifers inoculated with TK+ virus aborted between PI days 19 and 35. The finding of characteristic microscopic lesions and viral antigen in fetal tissues indicated that the abortions were caused by BHV-1 infection. Virus was isolated from 3 fetuses, and all isolates were TK+ virus. Two heifers inoculated with TK- bgal+ virus aborted at PI days 25 and 39. Fetal tissues had typical BHV-1 microscopic lesions and viral antigen. Virus was isolated from blood of both fetuses, and the isolates were TK- bgal+. Results of this study indicate that inactivation of the TK gene reduces, but does not eliminate, the abortifacient activity of BHV-1.     

Milk production and reproduction during a subclinical bovine herpesvirus 1 infection on a dairy farm

This study describes an outbreak of bovine herpesvirus 1 (BHV1) infections in a dairy herd with special reference to disease symptoms, reproductive performance and milk production losses. The study was carried out with a dairy herd consisting of 98 lactating animals. All animals were housed in the same freestall barn with intensive contact between all animals. An outbreak of BHV1 was induced by injecting three seropositive cows with dexamethasone. During the outbreak, no clinical signs were observed in any of the newly infected animals. At the time of infection, a significant drop in milk production was noted in animals that were initially-seronegative. The production loss was estimated at approximately 9.5 1 per infected animal during the infectious period of 14 days. None of the pregnant cows aborted because of BHV1 infection. During 50 days before BHV1 circulation, there was a significant decrease in the number of successful inseminations in both seronegative and seropositive animals. Therefore, it is doubtful that early pregnancies were terminated by BHV1 infection. The proportion of successful inseminations during the BHV1 circulation in this herd, and in the period thereafter, did not significantly differ from the baseline period.     

Detection of viral genome in non-neural tissues of cattle experimentally infected with bovine herpesvirus 1

Various bone disorders become manifest as cystic lesions. The differential diagnosis must include benign and malignant tumors and also non-tumorous lesions, such as osteomyelitis. The most important and most frequent types of genuine bone cyst are juvenile bone cyst and aneurysmal bone cyst. When juvenile bone cysts occur in adults they are called solitary bone cysts. Despite intensive research the pathogenesis of bone cysts is still unknown to this day, so that successful causal therapy is impossible. The main problem in the treatment of bone cysts is their high rate of recurrence, rates ranging between 20% and 50% having been cited in the international literature. A critical review of the literature reveals few publications with helpful follow-up results. Most of the publications are case reports, and they frequently merely describe various forms of treatment. More recent reports are mainly concerned with such methods as curettage, steroid injections, and continuous decompression with perforated screws. Until the early 1980s, segmental bone resection was the treatment of choice. Because of its high complication rate it has since been abandoned. In the last analysis, the only well-established method for which long-term results obtained in studies of any size have been published, is curettage of the cyst and grafting with cancellous bone from the iliac crest. In our series, 41 patients were treated with this method, and we recorded a recurrence rate of 17.1%. Complications were rare. The risk of recurrence depended on the age of the patient. A higher recurrence rate must be expected in children under the age of 10 years. For this reason, operative treatment should not be performed until after that age if possible. Newer methods, such as steorid injections and continuous decompression by means of perforated screws, had better results in some studies, but only according to a few authors. Further research is needed to show whether our method will yield good results in the long term when applied in larger patient collectives.     

Expression and cellular distribution of baculovirus-expressed bovine herpesvirus 1 (BHV-1) glycoprotein D (gD) sequences

Glycoprotein D (gD) of bovine herpesvirus 1 (BHV-1), a homolog of herpes simplex virus gD, represents a major component of the viral envelope and is a dominant immunogen. To study the antigenic properties of the different regions of gD, we have expressed the full-length gD encoding gene and overlapping fragments spanning various regions of the gD open reading frame in a baculovirus (Autographa californica nuclear polyhedrosis virus)--insect cell (Spodoptera frugiperda, SF-9) system. Maximum levels of expression for all proteins were obtained 48 to 72 h post infection of SF-9 cells by recombinant viruses. Full-length and truncated recombinant gD proteins reacted specifically with anti-gD monospecific serum as determined by immunoprecipitation and immunoblotting, indicating that the proteins retained their antigenicity. However, based on the reactivity with a panel of gD-specific monoclonal antibodies (Mabs), the full-length recombinant gD lacked proper expression for two highly neutralizing linear epitopes identified by Mabs R54 and 9D6. The rest of the epitopes appeared to be preserved and antigenically unaltered. Immunofluorescence studies of recombinant baculovirus infected SF-9 cells using gD monospecific serum, revealed no direct correlation between cellular localization of the expressed proteins and their amino acid sequences.     

Histamine causes Ca2+ entry via both a store-operated and a store-independent pathway in bovine adrenal chromaffin cells

The characteristics and properties of the increase in cytosolic [Ca2+] that occurs in bovine adrenal medullary chromaffin cells on exposure to histamine have been investigated. Specifically, these experiments were conducted to determine how much external Ca2+ enters the cell through a (capacitative) Ca2+ entry pathway activated as a consequence of intracellular Ca2+ store mobilization, relative to that which enters independently of store depletion via other channels activated by histamine. In Fura-2 loaded cells continued exposure to histamine (10 microM) caused a rapid but transient increase in cytosolic [Ca2+] followed by a lower plateau that was sustained as long as external Ca2+ was present. In the absence of external Ca2+, only the initial brief transient was observed. In cells previously treated with thapsigargin (100 nM) in Ca(2+)-free medium to deplete the internal Ca2+ stores, histamine caused no increase in cytosolic [Ca2+] when external Ca2+ was absent. Re-introduction of external Ca2+ to thapsigargin-treated store-depleted cells caused a sustained increase in cytosolic [Ca2+] that was further increased (P < 0.0002) upon exposure to histamine. The histamine-evoked increase was prevented by the H1-receptor antagonist, mepyramine (2 microM). A comparison was made between store-dependent Ca2+ entry consequent upon store mobilization with histamine in Ca(2+)-free medium and plateau phase Ca2+ entry resulting from stimulation with histamine in Ca(2+)-containing medium. The latter was found to be approximately 3 times greater in magnitude than the former (P < 0.0001) at the same concentration of histamine (10 microM). It is concluded that histamine causes Ca2+ entry not only via a capacitative entry pathway secondary to internal store mobilization, but also causes substantial Ca2+ entry through other pathways.     

Herpes simplex virus glycoprotein D can bind to poliovirus receptor-related protein 1 or herpesvirus entry mediator, two structurally unrelated mediators of virus entry

Several cell membrane proteins have been identified as herpes simplex virus (HSV) entry mediators (Hve). HveA (formerly HVEM) is a member of the tumor necrosis factor receptor family, whereas the poliovirus receptor-related proteins 1 and 2 (PRR1 and PRR2, renamed HveC and HveB) belong to the immunoglobulin superfamily. Here we show that a truncated form of HveC directly binds to HSV glycoprotein D (gD) in solution and at the surface of virions. This interaction is dependent on the native conformation of gD but independent of its N-linked glycosylation. Complex formation between soluble gD and HveC appears to involve one or two gD molecules for one HveC protein. Since HveA also mediates HSV entry by interacting with gD, we compared both structurally unrelated receptors for their binding to gD. Analyses of several gD variants indicated that structure and accessibility of the N-terminal domain of gD, essential for HveA binding, was not necessary for HveC interaction. Mutations in functional regions II, III, and IV of gD had similar effects on binding to either HveC or HveA. Competition assays with neutralizing anti-gD monoclonal antibodies (MAbs) showed that MAbs from group Ib prevented HveC and HveA binding to virions. However, group Ia MAbs blocked HveC but not HveA binding, and conversely, group VII MAbs blocked HveA but not HveC binding. Thus, we propose that HSV entry can be mediated by two structurally unrelated gD receptors through related but not identical binding with gD.     

Efficacy of a temperature-sensitive modified-live bovine herpesvirus type-1 vaccine against abortion and stillbirth in pregnant heifers

OBJECTIVE: To evaluate the efficacy of a commercially available temperature-sensitive modified-live bovine herpesvirus type-1 (BHV-1) vaccine against BHV-1 challenge-induced abortion and stillbirth. DESIGN: Prospective randomized control trial. ANIMALS: 20 cycling, nonpregnant, BHV-1 seronegative heifers of various breeds and weights, 12 to 15 months old. PROCEDURE: Heifers were randomly assigned to a vaccinate (n = 10) or nonvaccinate control (n = 10) group. Seventeen to 26 days after members of the vaccinate group received a second dose of vaccine, all heifers were artificially inseminated. Heifers were challenged intravenously with Cooper strain BHV-1 between days 177 and 187 of gestation. Aborted fetuses and stillborn calves were necropsied, and tissues collected for histologic examination and virus isolation. Heifers, calves, and fetuses were tested for BHV-1 antibody throughout the study. RESULTS: The difference in number of abortions or stillbirths between vaccinated heifers (1/10) and control heifers (10/10) was significant (P < 0.003). Seven of 10 control heifers had a virus neutralization antibody titer to BHV-1 at abortion or stillbirth that declined or remained unchanged from their titer at a previous serologic evaluation (7 to 66 days earlier). CLINICAL IMPLICATIONS: Prebreeding vaccination of replacement heifers with modified-live BHV-1 vaccine provides fetal protection at 6 months of gestation (7 months after vaccination) and appears to be a reasonable precaution to control economic losses associated with BHV-1 infection. Abortions induced by BHV-1 are not necessarily associated with rising or markedly high virus neutralization antibody titers. These titers should be used cautiously when assessing the role of BHV-1 in bovine abortion and stillbirth.     

A European inter-laboratory trial to evaluate the reliability of serological diagnosis of bovine herpesvirus 1 infections

Selective suppression of synaptic transmission during learning is proposed as a physiological mechanism for combining associative memory function at feedback synapses with self-organization of feedforward synapses in neocortical structures. A computational model demonstrates how selective suppression of feedback transmission allows this combination of synaptic function. During learning, sensory stimuli and the desired response are simultaneously presented as input to the network. Feedforward connections form self-organized representations of input, while suppressed feedback connections learn the transpose of the feedforward connectivity. During recall, suppression of transmission is removed, input activates the self-organized representation, and activity settles into a learned solution to the problem. This computational model can be used for learning of problems which are not linearly separable, including the negative patterning task (the XOR problem). Experiments in brain slice preparations of the rat somatosensory cortex tested whether the combination of self-organization and associative memory function could be provided by cholinergic suppression selective for feedback versus feedforward synapses. The cholinergic agonist carbachol selectively suppressed synaptic potentials elicited by stimulation of layer I (which contains a high percentage of feedback synapses), while having no effect on synaptic potentials elicited by stimulation of layer IV (with a high percentage of afferent and feedforward synapses).     

Functional complementation of UL3.5-negative pseudorabies virus by the bovine herpesvirus 1 UL3.5 homolog

The UL3.5 gene is positionally conserved but highly variable in size and sequence in different members of the Alphaherpesvirinae and is absent from herpes simplex virus genomes. We have shown previously that the pseudorabies virus (PrV) UL3.5 gene encodes a nonstructural protein which is required for secondary envelopment of intracytoplasmic virus particles in the trans-Golgi region. In the absence of UL3.5 protein, naked nucleocapsids accumulate in the cytoplasm, release of infectious virions is drastically reduced, and plaque formation in cell culture is inhibited (W. Fuchs, B. G. Klupp, H. Granzow, H.-J. Rziha, and T. C. Mettenleiter, J. Virol. 70:3517-3527, 1996). To assay functional complementation by a heterologous herpesviral UL3.5 protein, the UL3.5 gene of bovine herpesvirus 1 (BHV-1) was inserted at two different sites within the genome of UL3.5-negative PrV. In cells infected with the PrV recombinants the BHV-1 UL3.5 gene product was identified as a 17-kDa protein which was identical in size to the UL3.5 protein detected in BHV-1-infected cells. Expression of BHV-1 UL3.5 compensated for the lack of PrV UL3.5, resulting in a ca. 1,000-fold increase in virus titer and restoration of plaque formation in cell culture. Also, the intracellular block in viral egress was resolved by the BHV-1 UL3.5 gene. We conclude that the UL3.5 proteins of PrV and BHV-1 are functionally related and are involved in a common step in the egress of alphaherpesviruses.     

Attachment but not penetration of bovine herpesvirus 1 is necessary to induce apoptosis in target cells

Bovine herpesvirus 1 (BHV-1) induces apoptotic cell death in bovine peripheral blood mononuclear cells and B-lymphoma cells. Using a BHV-1 glycoprotein H null mutant, we have demonstrated that although penetration of BHV-1 is not required, attachment of BHV-1 viral particles is essential for the induction of apoptosis.     

Epidemiological characteristics of bovine herpesvirus 1 infections determined by bulk milk testing of all Dutch dairy herds

Samples of bulk milk were taken from all 33,636 Dutch dairy herds in November 1994 and tested for the presence of bovine herpesvirus 1 (BHV-1) antibodies with a gB-blocking ELISA. Sixteen per cent of the herds had a negative BHV-1 status in the bulk milk. Farms with only dairy cows were 1.9 times more likely to have a negative or weakly positive BHV-1 status than herds which also had beef/veal animals. Farms in areas containing less than one herd/km2 were 1.5 times more likely to have a negative or weakly positive BHV-1 status than herds in areas with more than three herds/km2. Differences in numbers of animals per unit area were not significantly associated with BHV-1 status. The probability of herds having a negative or weakly positive BHV-1 status decreased linearly with herd size by a factor of 1.2 per 10 animals. The purchase of stock was significantly associated with a negative or weakly positive BHV-1 status, but there was an interaction between farm type and purchase of stock. For farms with both dairy and beef/veal animals there was a weak association between the purchase of stock and BHV-1 status. For pure dairy herds the probability of having a negative or weakly positive BHV-1 status decreased linearly with the numbers of purchased stock by a factor of 1.3 per 10 animals purchased.     

Replication of bovine herpesvirus type 4 in human cells in vitro

A reference strain (Movár 33/63) of bovine herpesvirus type 4 (BHV-4) was inoculated into 14 different human cell lines and five primary cell cultures representing various human tissues. BHV-4 replicated in two embryonic lung cell lines, MRC-5 and Wistar-38, and in a giant-cell glioblastoma cell culture. Cytopathic effect and intranuclear inclusion bodies were observed in these cells. PCR detected a 10,000-times-higher level of BHV-4 DNA. Titration of the supernatant indicated a 100-fold increase of infectious particles. Since this is the first bovine (human herpesvirus 8 and Epstein-Barr virus related) herpesvirus which replicates on human cells in vitro, the danger of possible human BHV-4 infection should not be ignored.