Assessment of BoHV-4-based vector vaccine intranasally administered in a hamster challenge model of lung disease.

Introduction: Bovine herpesvirus 4 (BoHV-4) is a bovine Rhadinovirus not associated with a specific pathological lesion or disease and experimentally employed as a viral vector vaccine. BoHV-4-based vector (BoHV-4-BV) has been shown to be effective in immunizing and protecting several animal species when systemically administrated through intramuscular, subcutaneous, intravenous, or intraperitoneal routes. However, whether BoHV-4-BV affords respiratory disease protection when administered intranasally has never been tested. Methods: In the present study, recombinant BoHV-4, BoHV-4-A-S-ΔRS-HA-ΔTK, was constructed to deliver an expression cassette for the SARS-CoV-2 spike glycoprotein, and its immunogenicity, as well as its capability to transduce cells of the respiratory tract, were tested in mice. The well-established COVID-19/Syrian hamster model was adopted to test the efficacy of intranasally administered BoHV-4-A-S-ΔRS-HA-ΔTK in protecting against a SARS-CoV-2 challenge. Results: The intranasal administration of BoHV-4-A-S-ΔRS-HA-ΔTK elicited protection against SARS-CoV-2, with improved clinical signs, including significant reductions in body weight loss, significant reductions in viral load in the trachea and lungs, and significant reductions in histopathologic lung lesions compared to BoHV-4-A-S-ΔRS-HA-ΔTK administered intramuscularly. Discussion: These results suggested that intranasal immunization with BoHV-4-BV induced protective immunity and that BoHV-4-BV could be a potential vaccine platform for the protection of other animal species against respiratory diseases.

Bovine Herpesvirus-4 Based Vaccine Provides Protective Immunity against Streptococcus suis Disease in a Rabbit Model.

Streptococcus suis (S. suis) is a bacterial pathogen of pigs that has a major animal health and economic impact on the pig industry. Bovine herpesvirus-4 (BoHV-4) is a new virus-based vaccine vector that has been used for the immunogenic delivery of antigens from a variety of pathogens. In the present study, two recombinant BoHV-4-based vectors were evaluated for their ability to induce immunity and protection against S. suis in a rabbit model. The GMD protein is a fusion protein consisting of multiple dominant B-cell epitopes ((B-cell dominant epitopes of GAPDH, MRP, and DLDH antigens) (BoHV-4/GMD)) and the second suilysin (SLY) (BoHV-4/SLY) from S. suis serotype 2 (SS2). Both GMD and SLY delivered by the BoHV-4 vectors were recognized by sera from SS2-infected rabbits. The vaccination of rabbits with the BoHV-4 vectors induced antibodies against SS2, as well as against additional S. suis serotypes, SS7 and SS9. However, sera from BoHV-4/GMD-vaccinated animals promoted a significant level of phagocytic activity by pulmonary alveolar macrophages (PAMs) against SS2, SS7, and SS9. In contrast, sera from rabbits immunized with BoHV-4/SLY induced PAM phagocytic activity against only SS2. In addition, BoHV-4 vaccines differed in the associated level of protection against lethal SS2 challenge, which ranged from high (71.4%) to low (12.5%) for BoHV-4/GMD and BoHV-4/SLY, respectively. These data suggest BoHV-4/GMD as a promising vaccine candidate against S. suis disease.

Characterization and phylogenetic analysis of bovine gammaherpesvirus 4 isolated in China, 2022.

Bovine gammaherpesvirus 4 (BoHV-4) is a common virus detected in bovine with respiratory disease worldwide. In this study, we identified and characterized a novel BoHV-4 strain, referred as HB-ZJK, in vaginal swabs collected from cattle in China, 2022. The long unique region (LUR) of HB-ZJK is 10,9811 bp in length. It shares 99.17% to 99.38% nucleotide identity to five BoHV-4 strains available in GenBank and the highest similarity was seen with BoHV-4V. test (JN133502.1) strain (99.38%). Mutations, insertions or deletions were observed mainly in HB-ZJK gB (ORF8), TK (ORF21), gH (ORF22), MCP (ORF25), PK (ORF36), gM (ORF39), and gL (ORF47) genes compared to its genomic coordinates. Phylogenetic analyses of gB and TK genes showed that HB-ZJK clustered with China 512 (2019), B6010 (2009), and J4034 (2009) strains, demonstrating that the isolated HB-ZJK belongs to genotype 1. This is the first report that has revealed a comprehensive genome profile of BoHV-4 strain in China. This study will provide foundation for epidemiological investigations of BoHV-4 and contribute to the molecular and pathogenic studies of BoHV-4.

Study of the dynamics of in vitro infection with bovine gammaherpesvirus type 4 and apoptosis markers in susceptible cells.

Bovine gammaherpesvirus type 4 (BoHV-4) shows tropism for the endometrium, in which it causes the death of epithelial and stroma cells. Despite having anti-apoptotic genes in its genome, experiments based on immortalized cell lines have shown that BoHV-4 induces cell death by apoptosis. In the present study, we evaluated BoHV-4 replication, pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) mitochondrial genes expression and chromatin condensation in bovine endometrium primary culture cells (BEC) and in the Madin Darby bovine kidney (MDBK) cell line. Results showed that BoHV-4 has a preference for replication in BEC cells over the MDBK cell line, demonstrated by the high viral titer that is consistent with the tropism of the virus. In BEC cells, chromatin condensation was consistent with the values of viral kinetics at the late stage of infection, accompanied with a balance in the mRNA levels of apoptotic mitochondrial proteins. As a consequence, in those cells viral transmission would be enhanced by inhibiting apoptosis in the early stage of virus proliferation, allowing the complete production of viral progeny, and then, the induction of apoptosis in late stages would allow neighboring cells infection. In MDBK cells replication kinetics was coincident with the up-regulation of Bcl-2, which suggests that the productive infection in MDBK is associated with a lytic phase of the virus or another cell death pathway (probably autophagy mechanism) at the late stage of infection. The results agree with the study of nuclear morphology, where a constant chromatin condensation was observed over time. It is clear that the documented BoHV-4 apoptotic responses observed in the cell lines studied above are not valid in cells from primary cultures. The data presented in this study suggest that BoHV-4 could induce apoptosis in BEC cells without a leading role of the mitochondria pathway. Further studies will be necessary to characterize in detail the programmed cell death pathways involved in BoHV-4 infection in the primary cell cultures evaluated.

Assessment of replication of bovine herpesvirus type 4 in human glioblastoma and breast cancer cells as a potential oncolytic virus.

Oncolytic viruses have been extensively used in cancer treatment due to their tropism, selective replication only in tumor cells, and possible synergic interaction with other therapeutics. Different researchers have demonstrated that bovine herpesvirus 4 (BoHV-4), a member of the gammaherpesviridae family, has oncolytic potential in some human-origin cancer cell lines like glioma through the selective replication strategy. Using four apoptosis detection methods, namely MTT, LDH, TUNEL, and Annexin V assays, we evaluated the apoptotic effect of BoHV-4 Movar33/63 reference strain along with a recombinant BoHV-4 expressing EGFP in U87 MG cells (human glioblastoma cell line), MDA MB-231 (human breast cancer cell line), and MCF10a (non-tumorigenic human mammary epithelial cell line). Our findings indicate that this virus can replicate and induce apoptosis in these cell lines and hinder in vitro proliferation in a dose-dependent manner. In conclusion, BoHV-4 has in vitro potential as a novel oncolytic virus in human cancer therapy. However, its replication potential in the MCF10a cells as a non-tumorigenic human mammary epithelial cell line is a concern in using this virus in cancer therapy, at least against human mammary tumors. Further studies must therefore be conducted to examine the specific apoptotic pathways induced by this virus to move on to further experiments.

Mining the Unmapped Reads in Bovine RNA-Seq Data Reveals the Prevalence of Bovine Herpes Virus-6 in European Dairy Cows and the Associated Changes in Their Phenotype and Leucocyte Transcriptome.

Microbial RNA is detectable in host samples by aligning unmapped reads from RNA sequencing against taxon reference sequences, generating a score proportional to the microbial load. An RNA-Seq data analysis showed that 83.5% of leukocyte samples from six dairy herds in different EU countries contained bovine herpes virus-6 (BoHV-6). Phenotypic data on milk production, metabolic function, and disease collected during their first 50 days in milk (DIM) were compared between cows with low (1-200 and n = 114) or high (201-1175 and n = 24) BoHV-6 scores. There were no differences in milk production parameters, but high score cows had numerically fewer incidences of clinical mastitis (4.2% vs. 12.2%) and uterine disease (54.5% vs. 62.7%). Their metabolic status was worse, based on measurements of IGF-1 and various metabolites in blood and milk. A comparison of the global leukocyte transcriptome between high and low BoHV-6 score cows at around 14 DIM yielded 485 differentially expressed genes (DEGs). The top pathway from Gene Ontology (GO) enrichment analysis was the immune system process. Down-regulated genes in the high BoHV-6 cows included those encoding proteins involved in viral detection (DDX6 and DDX58), interferon response, and E3 ubiquitin ligase activity. This suggested that BoHV-6 may largely evade viral detection and that it does not cause clinical disease in dairy cows.

Epidemiological study of the association between bovine gammaherpesvirus type 4 and reproductive disease in dairy cattle from northwestern Spain.

Bovine gammaherpesvirus 4 (BoHV-4) has controversially been related with cattle reproductive disease. In the present study we analyze the relationship between exposure to BoHV-4 and reproductive performance in dairy cattle from northwestern Spain. A total of 2022 sera from 50 farms were examined to detect anti-BoHV-4 antibodies. Herd and individual reproductive records were collected to analyze association with exposure to BoHV-4. In addition, 52 abortion cases were examined to detect BoHV-4 DNA. An individual seroprevalence of 66.6 % and a herd prevalence of 98 % were found. Exposure to BoHV-4 increased with age, particularly in individuals between 26-36 months old (OR = 2.7; CI 95 %: 1.2-5.0, compared to animals < 26 months). Seroprevalence was not associated with herd fertility and herd abortion rate, but seropositive animals between 26-36 months presented prolonged calving to fertilizing insemination intervals (HR: 1.4; CI 95 %: 1.2-2.0) as well as higher odds of an unsuccessful 1st insemination (OR: 2.5; CI 95 %: 1.2-5.0). In abortion cases, BoHV-4 DNA was found in 12 vaginal swabs from 5 farms but not in any fetal tissue. Our results reveal an endemic, high and widespread exposure to BoHV-4 among dairy cattle from NW Spain with a limited impact in the reproductive performance of herds. The significantly worse reproductive performance of seropositive animals of 26-36 months of age may be the consequence of the establishment of primo-infections when moving heifers to lactation lots. Our findings may be useful to understand the potential population impact of BoHV-4.

Genetic characterization of bovine herpesvirus 4 (BoHV-4) isolates from Argentine cattle suggests a complex evolutionary scenario.

Bovine herpevsirus 4 (BoHV-4) is a gammaherpesvirus that has been associated with different clinical conditions in cattle. In Argentina, BoHV-4 was detected in diverse bovine samples. The aim of this study was to analyze the genetic relationship of 48 field BoHV-4 strains isolated from cattle in Argentina. According to thymidine kinase (tk) gene sequences, BoHV-4 isolates belong to genotypes 1, 2 and 3. Phylogenetic analyses confirmed the presence of the three previously described viral genotypes. However, some of the studied isolates presented conflicting phylogenetic signals between the studied markers. This suggests a complex evolutionary background, that is a history of recombination, incomplete lineage sorting (deep coalescence) or a combination of these, which requires further study. These potential events make difficult the diagnosis of BoHV-4 from clinical samples of cattle and may pose a significant problem for the control of the virus in the herds.

Analysis of the anti-apoptotic v-Bcl2 and v-Flip genes and effect on in vitro programmed cell death of Argentinean isolates of bovine gammaherpesvirus 4 (BoHV-4).

Some viruses encode inhibitory factors of apoptosis during infection to prolong cell viability and then to achieve a higher production of viral progeny or facilitate persistent infections. There is evidence that some gammaherpesviruses, including BoHV-4, carry genes that can both inhibit or induce apoptosis. BoHV-4 possesses two genes (ORF16 and ORF71) that code for proteins with anti-apoptotic functions, such as v-Bcl2 and v-Flip, respectively. Thus, it is relevant to study BoHV-4 in relation to the modulation of apoptosis in infected cells as a strategy for persistence in the host. The objective of this work was to analyze whether variations in v-Flip and v- Bcl2 of six phylogenetically divergent Argentinean isolates of BoHV-4 can influence the capacity of these strains to induce apoptosis in cell cultures. In this study, variations were mainly detected in the v-Flip gene and protein of the BoHV-4 strains belonging to genotype 3. Thus, it is possible to infer that sequence variations could be associated with some BoHV-4 genotype. Induction of apoptosis was not a significant event for any of the genetically distinct local isolates of BoHV-4 and there was not an evident relationship between the variability of both genes with the apoptotic effect of the phylogenetically distinct strains.

An assessment of bovine herpes virus 4 as a causative agent in abortions and neonatal death.

Numerous viruses, including bovine viral diarrhoea virus (BVDV), bovine herpes virus 1 (BoHV-1) and bovine herpes virus 4 (BoHV-4), and other pathogens are the most common causes of reproductive disorders and are responsible for huge economic losses in livestock production. This study investigates the aetiological role of BoHV-4 in fertility problems such as abortions, stillbirth and birth with unviable calves. Retrospective samples from 38 animals, including 17 aborting cows, 17 aborted foetuses, three stillborn calves and one unviable newborn calf were analysed. The BoHV-4 genome was detected in 25 (65.7%) animals by polymerase chain reaction. In 14 of these infected animals, we detected co-infection with BVDV, while the co-presence of BoHV-1 was also detected in one animal. In addition to the high prevalence of BoHV-4 genome in materials related to fertility problems, isolation of BoHV-4 from the brain of one stillborn calf indicated a causal link between BoHV-4 and fertility problems, such as abortion, stillbirths or birth with unviable calves.

Monitoring of the shedding and serological dynamics of Bovine gammaherpesvirus type 4 in a dairy cattle herd.

Bovine gammaherpesvirus type 4 (BoHV-4) is increasingly related with reproductive disease in cattle, but its epidemiology is not fully understood. We monitored the serological response and shedding of BoHV-4 in a positive dairy cattle farm with metritis. First, we performed an ELISA to detect BoHV-4 antibodies in all the animals (n = 104). Afterwards, ten seronegative heifers introduced in the production lot and sera samples were monthly taken for four months and then 6-10 months after introduction to detect BoHV-4 antibodies by ELISA. Moreover, a vaginal swab was taken after calving to detect BoHV-4 by PCR. Concurrently, a weekly collection of vaginal and nasal swabs and milk was performed during the first month post-partum in multiparous cows with metritis (n = 14), heifers with metritis (n = 4), heifers without metritis but positive to BoHV-4 (ELISA or PCR) (n = 2) and multiparous cows without metritis (n = 3). Seropositivity was higher in older animals and in the production lot. Three heifers which shed BoHV-4 after parturition resulted seronegative at first but eventually seroconverted. In the same vein, most heifers seroconverted after 6-10 months in the production lot (8/10). Multiparous cows shed virus by various routes: 13/14 (93 %) in vaginal secretions, 7/14 (50 %) in nasal exudates and 7/14 (50 %) in milk. However, in the other groups, shedding was only detected in vaginal swabs from the first week post-partum. Our study describes BoHV-4 shedding in field conditions. Seronegative animals may become horizontally infected when moved to a contaminated environment.

Comparative analysis of replicative properties of phylogenetically divergent, Argentinean BoHV-4 strains in cell lines from different origins.

Bovine gammaherpesvirus 4 (BoHV4) is a member of the family Herpesviridae. In Argentina, BoHV4 was isolated and characterized in 2007 from samples of aborted cows. Argentinean isolates are highly divergent and are classified as: Genotype 1(Movar-like), Genotype 2 (DN599-like) and Genotype 3 (a novel group). The aim of this study was to comparatively evaluate the biological characteristics of six Argentinean BoHV4 field isolates in cell lines from different origins. All strains induced productive infection in the cell lines used, with different degrees of permissiveness. A direct relationship among the times of appearance of cytopathic effect, the growth kinetics, the size of the lysis plaques and the virulent-like behaviour in vitro could not be established. However, although slight, there are differences in the biological behaviour of the BoHV4 fields isolates analyzed. This variability is independent of their genetic classification but would be conditioned by the nature of the infected cells.

Presence of gammaherpesvirus BoHV-4 in endometrial cytology samples is not associated with subclinical endometritis diagnosed at artificial insemination in dairy cows.

In the past, bovine herpesvirus 4 (BoHV-4) has been suggested to be associated with metritis and endometritis. However, not many field studies investigated the association between BoHV-4 and subclinical endometritis (SCE). In the present study, the association between the intrauterine presence of BoHV-4 and SCE diagnosed during artificial insemination (AI) was examined on two dairy farms in Belgium. An immunoperoxidase monolayer assay (IPMA) and an enzyme-linked immuno sorbent assay (ELISA) were used to screen the serum for anti-BoHV-4 antibodies. A SYBR green based one step real time qPCR was used to detect and quantify BoHV-4 (ORF20) in nasal, uterine and vaginal samples collected at AI. A reverse transcription qPCR (RT-qPCR) was used to detect mRNA (gB) as proof of a productive BoHV-4 infection. BoHV-4 was detected in 39.4% (farm A)/23.8% (farm B) of the nasal samples, 48.5% (farm A)/19.0% (farm B) of the uterine samples and 51.5% (farm A)/42.9% (farm B) of the vaginal samples. Active replication was only detected in farm A in 38.5% of the BoHV-4 positive nasal samples and in 5.9% positive cases of the vaginal samples. The prevalence of SCE diagnosed at AI was 45.5% and 42.9% in farm A and farm B, respectively. The presence of SCE was associated with a reduced pregnancy outcome at artificial insemination (AI) (P＜0.001). The occurrence of SCE at AI was not associated with the presence of latent or productive BoHV4 infections in the uterus nor in the vagina and nose (P＞0.05).

Bovine Herpesvirus-4-Based Vector Delivering Peste des Petits Ruminants Virus Hemagglutinin ORF Induces both Neutralizing Antibodies and Cytotoxic T Cell Responses.

Peste des Petits Ruminants Virus (PPRV) is an extremely infective morbillivirus that primarily affects goats and sheep. In underdeveloped countries where livestock are the main economical resource, PPRV causes considerable economic losses. Protective live attenuated vaccines are currently available but they induce antibody responses similar to those produced in PPRV naturally infected animals. Effective vaccines able to distinguish between vaccinated and naturally infected animals are required to PPRV control and eradication programs. Hemagglutinin (H) is a highly immunogenic PPRV envelope glycoprotein displaying both hemagglutinin and neuraminidase activities, playing a crucial role in virus attachment and penetration. In this study, a recombinant Bovine Herpesvirus-4 (BoHV-4)-based vector delivering an optimized PPRV-Hemagglutinin expression cassette, BoHV-4-A-PPRV-H-ΔTK, was assessed in immunocompetent C57BL/6 mice. BoHV-4-A-PPRV-H-ΔTK-immunization elicited both cellular and humoral immune responses with specific T cell, cytotoxic T lymphocyte, and sero-neutralizing antibody against PPRV. These data suggest recombinant BoHV-4-A-PPRV-H-ΔTK as an effective vaccine candidate to protect against PPRV herd infection and potentially applicable for eradication programs.

Laboratory Findings Suggesting an Association Between BoHV-4 and Bovine Abortions in Southern Belgium.

Abortions cause heavy economic losses for the bovine sector. The use of a standardized panel of analyses covering a large spectrum of pathogens responsible of abortion in cattle allowed demonstrating the direct involvement of at least one pathogen in 57% of analysed abortions in the southern part of Belgium. This result suggests a margin of improvement in the diagnostic efficacy. In order to evaluate the interest to broaden the list of pathogens included in the panel of analyses, the implication of bovine herpesvirus 4 (BoHV-4) in abortion was assessed by two different studies. In the first study, coupled serology was performed after abortion on 714 dams to identify specific seroconversion against BoHV-4. The overall seroconversion in cows was 19.5%, with a higher frequency in primiparous compared to multiparous females. In addition, the type of breed (beef cattle) and the time period from the fourth quarter 2008 until the last quarter 2009 were significantly related to the seroconversion of cows. The second study investigated the virus ability to infect the foetus. In this study, 368 cases of bovine abortions were specifically tested for BoHV-4, using PCR on foetus tissues and ELISA on dam and foetus sera. The results showed a maternal seroprevalence of 64.7%, a foetal seroprevalence of 0.8% and a PCR prevalence in foetuses of 1.1%, demonstrating the ability of BoHV-4 to infect the foetus.

Bovine herpesvirus 4-based vector delivering a hybrid rat/human HER-2 oncoantigen efficiently protects mice from autochthonous Her-2+ mammary cancer.

The epidermal growth factor receptor 2 (HER-2) oncogene is a major target for the immunotherapy of breast cancer. Following up to the therapeutic success achieved with Her-2-targeting monoclonal antibodies, immune-prophylactic approaches directed against Her-2 have also been investigated taking into account, and trying to overcome, Her-2 self-tolerance. Perhaps due to safety (and efficacy) concerns, the least explored anti-Her-2 active immunization strategy so far has been the one relying on viral-vectored vaccine formulations. Taking advantage of the favorable properties of bovine herpesvirus 4 (BoHV-4) in terms of safety and ease of manipulation as well as its previously documented ability to transduce and confer immunogenicity to heterologous antigens, we tested the ability of different recombinant HER-2-BoHV-4 immunogens to 8break tolerance and elicit a protective, anti-mammary tumor antibody response in HER-2 transgenic BALB-neuT mice. All the tested constructs expressed the HER-2 transgenes at high levels and elicited significant cellular immune responses in BALB/c mice upon administration via either DNA vaccination or viral infection. In BALB-neuT mice, instead, only the viral construct expressing the membrane-bound chimeric form of Her-2 protein (BoHV-4-RHuT-gD) elicited a humoral immune response that was more intense and earlier-appearing than that induced by DNA vaccination. In keeping with this observation, two administrations of BoHV-4-RHuT-gD effectively protected BALB-neuT mice from tumor formation, with 50% of vaccinated animals tumor-free after 30 weeks from immunization compared to 100% of animals exhibiting at least one palpable tumor in the case of animals vaccinated with the other BoHV-4-HER-2 constructs.

Development and Evaluation of an Indirect ELISA: Serological Survey to Detect Specific Antibodies to Bovine Herpesvirus 4

ABSTRACTThe aim of this work was to develop and evaluate an indirect enzyme-linked immunosorbent assay (ELISA) and made a serological screening for specific antibodies to BoHV-4. Bovine serum samples were collected from different Brazilian states and evaluated for the presence of antibodies for BoHV-4 and BoHV-1. The serological results obtained showed that the indirect ELISA assay could be applied for the detection of specific antibodies for BoHV-4. The ELISA test allowed concluding that BoHV-4 is present in bovines in every Brazilian state from which serum samples were collected. The ELISA assay here standardized proved to be useful for the epidemiological studies and showed a positivity range from 1.8 to 66% in Brazil.

Bovine Herpesvirus 4 in Parana State, Brazil: case report, viral isolation, and molecular identification.

Bovine Herpesvirus 4 (BoHV-4) is a member of Gammaherpesvirinae sub-family and belongs to genus Rhadinovirus . This virus has been associated with different clinical manifestations and research activity has put forward a strong correlation among virus infection, postpartum metritis, and abortion. The goal of this work was to characterize a virus strain isolate from a cow's uterine outflow. From swabs drawn of uterine secretion, a virus strain was isolated and characterized by its cytopathology, morphology, and molecular biology approaches. In culture there was CPE development, characterized mainly by long strands with several small balloons along them, radiated from infected cells. Electron microscopy analysis revealed virus particles that had icosahedrical capsid symmetry surrounded by a loose envelope, typical of a herpesvirus. A 2,571 bp PCR product after Hind III digestion generated four fragments, whose base pair composition were 403, 420, 535, and 1,125 bp. Restriction enzymes Hind III and Bam HI generated the expected diagnostic bands as well as a 2,350 bp hypermolar fragment as a result of Bam HI treatment to demonstrate that agent was a bovine herpesvirus 4, appertaining to DN-599 group.

Bovine Herpesvirus 4 in Parana State, Brazil: case report, viral isolation, and molecular identification

Bovine Herpesvirus 4 (BoHV-4) is a member of Gammaherpesvirinae sub-family and belongs to genus Rhadinovirus. This virus has been associated with different clinical manifestations and research activity has put forward a strong correlation among virus infection, postpartum metritis, and abortion. The goal of this work was to characterize a virus strain isolate from a cow’s uterine outflow. From swabs drawn of uterine secretion, a virus strain was isolated and characterized by its cytopathology, morphology, and molecular biology approaches. In culture there was CPE development, characterized mainly by long strands with several small balloons along them, radiated from infected cells. Electron microscopy analysis revealed virus particles that had icosahedrical capsid symmetry surrounded by a loose envelope, typical of a herpesvirus. A 2,571 bp PCR product after HindIII digestion generated four fragments, whose base pair composition were 403, 420, 535, and 1,125 bp. Restriction enzymes HindIII and BamHI generated the expected diagnostic bands as well as a 2,350 bp hypermolar fragment as a result of BamHI treatment to demonstrate that agent was a bovine herpesvirus 4, appertaining to DN-599 group.

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.