Bovine herpesvirus 5 BICP0 complements the bovine herpesvirus 1 homolog

Bovine herpesvirus 1 (BoHV-1) and BoHV-5 are closely related (82% amino acid identity) but differ strongly in neuropathogenesis. The immediate-early gene for BICP0 is less conserved (70% amino acid identity) and may contribute to a dissimilar phenotype. A peculiar difference is a guanosine hexamer in the BICP0-1 gene which aligns with only five guanosines in the BICP0-5 gene and therefore results in a frameshift in the latter open reading frame. Thus, the C-terminal amino acid sequence (residues 643–676 of BICP0-1 vs. 655–720 of BICP0-5) is completely different. We introduced the BICP0-5 frameshift into the BoHV-1 genome cloned as a bacterial artificial chromosome (BoHV-1 BAC) using the Red recombination system with galK selection and counterselection. Transfection of MDBK cells with the resulting BAC produced recombinant virus that replicated like wild type BoHV-1 in vitro. Attempts to exchange the entire BICP0-1 gene by the BoHV-5 homolog using the same approach failed repeatedly. Therefore, we cotransfected purified BICP0⁻/galK⁺-BoHV-1 BAC DNA with a recombination plasmid coding for BICP0-5 with or without a HA tag into MDBK cells. BoHV-1 recombinants expressing the respective proteins were characterized. In vitro, all recombinants grew to similar titers as the parental viruses, which demonstrates that BICP0-5 compensates for the growth defect of BICP0⁻/galK⁺-BoHV-1 and functionally complements BICP0-1 of BoHV-1. We conclude that BICP0 may be suitable to positively select BoHV-1 recombinants with deletions or insertions of additional genes of interest.     

Experimental infection of calves with a gI, gE, US9 negative bovine herpesvirus type 5

In this work, a role for the genes encoding glycoproteins I (gI) and E (gE) and the US9 protein of bovine herpesvirus type 5 (BHV-5) in neuropathogenicity and reactivation of latent infections was examined. Calves infected intranasally with a gI/gE/US9 deleted recombinant shed up to 10(2.85) TCID50/ml infectious virus in nasal secretions. Calves infected with the wild type BHV-5 parental virus shed up to 10(5) TCID50/ml virus. No signs of disease were observed in calves infected with the recombinant virus, whereas those infected with wild type virus displayed respiratory and neurological signs. The recombinant was only able to reach the basal portions of the central nervous system. In contrast, wild type virus was found widespread within the brain. Reactivation with dexamethasone 60 days post-infection resulted in reactivation of wild type virus, whereas the recombinant virus could not be reactivated. These studies demonstrate that genes gI, gE and US9 of BHV-5 are important for its neuropathogenicity and its ability to reactive from latency.     

Studies on the pathogenicity of bovine herpesvirus type 5 in sheep.

Four Merino lambs were intranasally inoculated with bovine herpesvirus type 5 (BHV-5) reference strain N569. Two lambs were mock-inoculated as negative controls. The virus-inoculated animals developed apathy, inappetence, rhinitis, nasal, ocular and genital discharge, slight diarrhea and neurological disorders, like tremor and salivation. BHV-5 was isolated from the nasal discharge in two of the animals, while the polymerase chain reaction (PCR) detected the virus in all the infected lambs. Two lambs died on post infection day (PID) 13, while the other two infected animals were euthanized on PID 15 and 30. Gross pathological changes were not observed, however, histopathological examinations revealed diffuse nonsuppurative meningo-encephalitis in all infected animals. Viral antigen was detected by immunohistochemistry and viral nucleic acid was revealed by in situ hybridization in the brain of the two lambs, which died on PID 13. The virus was demonstrated by virus isolation and by PCR from different organs of all the infected animals. Slight rise of antibodies was observed in the infected animals from PID 15. The results show that BHV-5 is able to cross the species barrier and may establish infection in sheep.     

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.     

Effect of dexamethasone on bovine leukocyte functions and bovine herpesvirus type-1 replication.

In an attempt to elucidate the mechanism whereby dexamethasone could reactivate bovine herpesvirus type-1 the effect of dexamethasone on virus replication and leukocyte functions was assessed. No effect was detectable on either virus yield or in vitro replication kinetics. In contrast, dexamethasone influenced several leukocyte functions thought to be of importance in antiviral defense and maintenance of latency. In vitro exposure of peripheral blood polymorphonuclear neutrophilic granulocytes of normal animals to dexamethasone depressed their migratory and cytotoxic activities, but had no effect on Fc- and complement receptor expression. Dexamethasone also depressed lectin-induced lymphocyte proliferation and interleukin-2 generation in a dose-dependent manner. When cows were treated repeatedly with dexamethasone and their leukocytes assayed, suppression of phytohemagglutinin-induced lymphocyte proliferation, interleukin-2 generation, natural cytotoxicity of mononuclear cells and polymorphonuclear neutrophilic granulocyte functions were observed. In contrast, concanavalin A induced lymphocyte proliferation was increased following treatment.     

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.     

Primary infection,latency, and reactivation of bovine herpesvirus type 5 in the bovine nervous system

Bovine herpesvirus type 5 (BHV-5) infection in calves causes meningoencephalitis, a fatal disease highly prevalent in South America. To study the pathogenesis of BHV-5 infection in cattle, 12 calves (group 1: acute infection) and 11 calves (group 2: latent infection) were intranasally inoculated with an Argentinean BHV-5 isolate at 10(8) and 10(4.7) tissue culture infective doses, respectively; six calves (control group) were mock infected. At 3 months postinoculation, all of the calves in group 2 and three calves in group 3 were given dexamethasone to reactivate the virus. The animals were euthanatized between days 6 and 17 postinoculation (group 1) and between days 6 and 16 postreactivation (group 2). Seventy-five percent and 91% of animals in groups 1 and 2, respectively, excreted BHV-5 in nasal and ocular discharges. Following dexamethasone administration, 45% of calves shed virus in both types of secretions. Spontaneous virus reactivation and shedding was observed in one calf. Neurologic signs consisting of circling, teeth grinding, ptyalism, jaw chomping, tongue protrusion, and apathy were observed in two animals in group 1 and, during the reactivation period, in four animals in group 2. Macroscopic findings consisted of softening of the cerebral tissue, meningeal hemorrhages and swelling, and edema and hemorrhages of prescapular, retropharyngeal and submandibular lymph nodes. Histologic lesions consisted of meningitis, mononuclear perivascular cuffing, neuronophagia, satellitosis, gliosis, hemorrhage, and necrosis and edema. Lesions in anterior cerebral cortex, medulla, and pons were consistently seen in all the animals of group 1. In the acutely infected animals, lesions in the diencephalon appeared at day 10 postinoculation, whereas in the latently infected calves these lesions were observed as early as at day 6 postreactivation. Latently infected animals developed lesions simultaneously in anterior cortex, medulla, pons, and diencephalon, showing a remarkable difference from the acutely infected group. Trigeminal ganglionitis appeared relatively early in animals of both groups (day 7 postinoculation in group 1 and day 8 postreactivation in group 2).     

Immunological relationships between malignant catarrhal fever virus (alcelaphine herpesvirus 1) and bovine cytomegalovirus (bovine herpesvirus 3)

Strains of malignant catarrhal fever virus (alcelaphine herpesvirus 1 (AHV-1)) and bovine cytomegalovirus (bovine herpesvirus 3 (BHV-3)) were compared for serological relatedness by cross-titration in an indirect immunofluorescent (IIF) antibody assay. There was definite cross-reactivity between these 2 viruses, with heterologous sera staining intracellular and membrane antigens of infected cells. Heterologous antibody titres were approximately 50-fold lower than homologous titres and could be removed by absorption with either homologous or heterologous virus-infected cells, but not with uninfected cells. Regression analyses of IIF antibody titres to AHV-1 and BHV-3 virus in 3 groups of wild ungulate sera also indicated a serological relationship between these herpesviruses. In a cross-immunity trial, 2 of 3 cattle immunized with a BHV-3 virus and 2 of 3 cattle immunized with avirulent AHV-1 resisted challenge with virulent AHV-1-infected blood which killed 3 unimmunized controls. These results are discussed particularly with respect to the involvement of BHV-3 in malignant catarrhal fever.     

Development of a polymerase chain reaction and restriction typing assay for the diagnosis of bovine herpesvirus 1, bovine herpesvirus 2, and bovine herpesvirus 4 infections.

A multiplex polymerase chain reaction (PCR) method coupled with a restriction analysis of PCR products (PCR with restriction fragment length polymorphism) was developed for the simultaneous detection of bovine herpesvirus 1, bovine herpesvirus 2, and bovine herpesvirus 4 infections. The specificity, sensitivity, and practical diagnostic applicability of this method were evaluated. This assay may be also adapted to the diagnosis of suid herpesvirus 1 and equine herpesviruses 1 and 3 and could become a powerful diagnostic tool.     

Susceptibility of mice to bovine herpesvirus type 5 infection in the central nervous system

Bovine herpesvirus type 5 (BoHV-5) is an important pathogen that causes meningoencephalitis in cattle. Few studies have used the mouse as a model for BoHV-5 infection. Despite the fact that BoHV-5 can infect mice with immune deficiencies, little is known about viral replication, immune response, and the course of infection in the central nervous system (CNS) of wild-type mice. Therefore, the aim of this study was to evaluate the response in the CNS of BALB/c mice acutely infected with BoHV-5 at different days post-inoculation (dpi). BoHV-5, when inoculated intracranially, was able to infect and replicate within the CNS of BALB/c mice. Until 15 dpi, the mice were able to survive without showing prominent neurological signs. The infection was accompanied by a Th1 immune response, with a significant expression of the cytokines IFN-γ and TNF-α and chemokine CCL-2. The expression of these cytokines and chemokines was most significant in the early course of infection (3 and 4 dpi), and it was followed by meningoencephalitis with perivascular cuffing and periventriculitis, composed mainly of macrophages and lymphocytes. After the expression of cytokines and chemokine, the mice were able to curb BoHV-5 acute infection in the brain, since there was a decrease in the number of BoHV-5 DNA copies after 3 dpi and viable viral particles were not detected after 6 dpi. Importantly, BoHV-5 was able to infect the trigeminal ganglia during acute infection, since a large number of BoHV-5 DNA copies were detected on 1 and 2 dpi.     

Efficacy of an inactivated,recombinant bovine herpesvirus type 5 (BoHV-5) vaccine

Bovine herpesvirus type 5 (BoHV-5) is the causative agent of bovine herpetic encephalitis. In countries where BoHV-5 is prevalent, attempts to vaccinate cattle to prevent clinical signs from BoHV-5-induced disease have relied essentially on vaccination with BoHV-1 vaccines. However, such practice has been shown not to confer full protection to BoHV-5 challenge. In the present study, an inactivated, oil adjuvanted vaccine prepared with a recombinant BoHV-5 from which the genes coding for glycoprotein I (gI), glycoprotein E (gE) and membrane protein US9 were deleted (BoHV-5 gI/gE/US9⁻), was evaluated in cattle in a vaccination/challenge experiment. The vaccine was prepared from a virus suspension containing a pre-inactivation antigenic mass equivalent to 10^7.69 TCID₅₀/dose. Three mL of the inactivated vaccine were administered subcutaneously to eight calves serologically negative for BoHV-5 (vaccinated group). Four other calves were mock-vaccinated with an equivalent preparation without viral antigens (control group). Both groups were boostered 28 days later. Neither clinical signs of disease nor adverse effects were observed during or after vaccination. A specific serological response, revealed by the development of neutralizing antibodies, was detected in all vaccinated animals after the first dose of vaccine, whereas control animals remained seronegative. Calves were subsequently challenged on day 77 post-vaccination (pv) with 10^9.25 TCID₅₀ of the wild-type BoHV-5 (parental strain EVI 88/95). After challenge, vaccinated cattle displayed mild signs of respiratory disease, whereas the control group developed respiratory disease and severe encephalitis, which led to culling of 2/4 calves. Searches for viral DNA in the central nervous system (CNS) of vaccinated calves indicated that wild-type BoHV-5 did not replicate, whereas in CNS tissues of calves on the control group, viral DNA was widely distributed. BoHV-5 shedding in nasal secretions was significantly lower in vaccinated calves than in the control group on days 2, 3, 4 and 6 post-challenge (pc). In addition, the duration of virus shedding was significantly shorter in the vaccinated (7 days) than in controls (12 days). Attempts to reactivate latent infection by administration of dexamethasone at 147 days pv led to recrudescence of mild signs of respiratory disease in both vaccinated and control groups. Infectious virus shedding in nasal secretions was detected at reactivation and was significantly lower in vaccinated cattle than in controls on days 11–13 post-reactivation (pr). It is concluded that the inactivated vaccine prepared with the BoHV-5 gI/gE/US9⁻ recombinant was capable of conferring protection to encephalitis when vaccinated cattle were challenged with a large infectious dose of the parental wild type BoHV-5. However, it did not avoid the establishment of latency nor impeded dexamethasone-induced reactivation of the virus, despite a significant reduction in virus shedding after challenge and at reactivation on vaccinated calves.     

Acute and latent infection by bovine herpesvirus type 5 in experimentally infected goats

The ability of alphaherpesviruses to infect different ruminant species may have important implications for control/eradication efforts. Serological data indicate that goats may be naturally infected with bovine herpesviruses. To investigate the susceptibility of goats to bovine herpesvirus-5 (BoHV-5), 3-4-month-old kids were inoculated intranasally with each of three Brazilian BoHV-5 isolates (G1, n=8; G2, n=5; G3, n=5). The acute infection was characterized by virus shedding in nasal secretions for up to 14 days (titers up to 10(5.97)TCID(50)/mL), mild respiratory signs and conjunctivitis. All animals seroconverted to BoHV-5, developing virus neutralizing (VN) titers from 4 to 32 to the homologous viruses. At day 60 post inoculation (pi), two animals from each group were euthanized for tissue collection and the remaining goats were submitted to dexamethasone administration (0.4 mg kg(-1) for 5 days). Dexamethasone treatment resulted in virus reactivation in 9 out of 12 animals, as ascertained by virus shedding and/or by increase in VN titers. Virus shedding was detected in 8/12 animals and lasted from 1 to 9 days. Latent viral DNA was detected by PCR in the olfactory bulb and/or trigeminal ganglia of 6/6 goats euthanized at day 60 pi and in 12/12 animals euthanized 40 days post-dexamethasone. These results show that young goats are susceptible to BoHV-5 and may shed virus upon reactivation of latent infection. Thus, it is reasonable to expect that goats raised in close contact with cattle in areas where BoHV-5 is endemic may be infected and therefore should be considered potential reservoirs of the virus.     

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.     

Isolation of bovine herpesvirus type 5 from the semen of a healthy bull in Australia

Artificial insemination is widely used in the cattle industry and a major challenge is to ensure that semen is free of infectious agents. A healthy donor bull was tested for freedom from infectious agents. A bovine herpesvirus was isolated in testis cells and identified as bovine herpesvirus type 5 (BoHV-5) by polymerase chain reaction and by direct amplicon sequencing. The amplicon sequence shared 100% similarity with the published sequence of BoHV-5. This is the first report in Australia of BoHV-5 in semen. The implications of this finding are discussed.     

人端粒酶基因(hTERT)对牛耳成纤维细胞凋亡的影响

为了提高体细胞核移植的效率,试验探讨了转染外源性hTERT基因对核移植供体细胞———牛耳成纤维细胞的影响。试验应用脂质体介导hTERT基因转染,用相差显微镜观察转染前后的细胞状态,用RT-PCR、W estern blot法检测hTERT基因是否整合并表达,用流式细胞仪检测转染前后细胞周期的变化和凋亡的情况,并进行核型分析。结果表明:转染48 h后能看到明显的荧光,经600μg/mLG418筛选后,获得阳性克隆,转染后的细胞形态上基本无变化,hTERT基因整合并表达;经流式细胞仪检测,转染前后生长周期无明显变化,凋亡率极显著降低(P<0.01),核型正常;转染hTERT基因后的牛耳成纤维细胞的凋亡率显著低于未转染的细胞,并且形态正常,表明转染hTERT后,使体外培养的牛耳成纤维细胞生命力得到延长,为核移植提供了数量更多的高质量的核供体细胞。     

CPEB3 regulates the proliferation and apoptosis of bovine cumulus cells

Cytoplasmic polyadenylation element‐binding protein 3 (CPEB3) is a member of the Cytoplasmic polyadenylation element‐binding family, which has been found to regulate the translation of dormant and masked mRNA in Xenopus oocytes and plays potential roles in regulating biological functions in cells and tissues. However, its role in cumulus cells is not clear. In this study, the mRNA expression of CPEB3 in bovine cumulus cells was inhibited with small interfering RNA. Cell cycle progression, proliferation, and apoptosis were measured after inhibition of CPEB3. Subsequently, changes in intracellular Reactive oxygen species content, mitochondrial membrane potential and expansion‐related gene expression were examined. The results showed that after CPEB3 inhibition, cumulus cells had an abnormal cell cycle, the numbers of cells in the S and G2/M phases were significantly increased, cell proliferation was increased and apoptosis rates were decreased. These effects were likely due CPEB3 inhibition‐induced decreases in intracellular Reactive oxygen species levels; increases in mitochondrial membrane potential; decreases in apoptosis; downregulation of CCNA, CCND, CCNE, CDK2, CDK4, CDK6, p21, and p27 mRNA expression; and upregulation of CCNB, CDK1, HAS2, PTGS2, PTX3, and CEBPB mRNA expression. Therefore, CPEB3 plays potential roles in regulating the biological and physiological functions of bovine cumulus cell.     

Experimental infection of sheep with bovine herpesvirus type-5 (BHV-5): acute and latent infection

We demonstrated that sheep are susceptible to acute and latent infection by bovine herpesvirus type-5 (BHV-5). Lambs inoculated intranasally with two South American BHV-5 isolates replicated the virus with titers up to 10(7.1) TCID50/ml for up to 15 days and showed mild signs of rhinitis. Four lambs in contact with the inoculated animals acquired the infection and excreted virus for up to seven days. One lamb developed progressive signs of neurological disease and was euthanized in extremis. Clinical signs consisted of tremors of the face, bruxism, ptyalism, incoordination, lateral flexion of the neck and head, circling, walking backwards, recumbency and paddling. The virus was detected in the anterior and posterior cerebrum, dorso- and ventro-lateral cortex, cerebellum, pons, midbrain and olfactory bulb. Viral nucleic acids were demonstrated in neurons and astrocytes of the anterior and ventro-lateral cortex by in situ hybridization. Histological changes consisting of non-suppurative meningitis, perivascular mononuclear cuffing, focal gliosis, neuronal necrosis and intranuclear inclusions were observed in the anterior cerebrum, ventro-lateral cortex and midbrain. Dexamethasone treatment at Day 50 pi resulted in reactivation of the latent infection and virus shedding in 13/16 (81%) of the lambs. Together with previous reports of BHV-5 antibodies in sheep, these findings show that sheep are fully susceptible to BHV-5 suggesting that infection by BHV-5 in sheep may occur naturally.     

Effect of bovine adenovirus 3 on mitochondria

Viruses alter the structure and the function of mitochondria for survival. Electron microscopy analysis of the cells infected with bovine adenovirus 3 revealed extensive damage to the inner mitochondrial membrane characterized by dissolution of the cristae and amorphous appearance of mitochondrial matrix with little or no damage to the outer mitochondrial membrane. There were fewer cristae with altered morphology. Potential patches of protein synthesis machinary around mitochondria could be observed at 12 hours post infection (hpi). At 24 hpi, the multi vascular bodies were evident throughout the infected cell. ATP production, mitochondrial Ca²⁺ and mitochondrial membrane potential (MMP) peaked at 18 hpi but decreased significantly at 24 hpi. This decrease coincided with the increased production of superoxide (SO) and reactive oxygen species (ROS), at 24 hpi indicating acute oxidative stress in the cells and suggesting a complete failure of the cellular homeostatic machinary. The results reveal an intericate relationship between Ca²⁺ homeostasis, the ATP generation ability of cells, SO and ROS production, and regulation of MMP following infection by bovine adenovirus 3.