Detection of Bovine herpesvirus 4 DNA in aborted bovine fetuses

The presence of Bovine herpesvirus 4 (BoHV-4) was investigated by several methods in 24 aborted bovine fetuses. Polymerase chain reaction (PCR) and in situ DNA hybridization proved the presence of BoHV-4 DNA in 7 (29%) of the fetuses. The BoHV-4 genome was detected in the cytoplasm of splenic lymphocytes and monocytes, and sometimes in renal tubular epithelial cells or hepatic Kupffer cells, in all 7 PCR-positive fetuses. However, BoHV-4-specific monoclonal antibody failed to detect viral antigen in the formalin-fixed, paraffin-embedded tissue samples. No bacterial pathogens were found in the tissues of the BoHV-4-positive fetuses. Fungi were detected in 1 sample, and antibody to bovine viral diarrhea virus was detected in another. These results indicate that BoHV-4 could play a role in reproductive disorders of cattle, including abortion.     

Bovine herpesvirus type 4: a special herpesvirus (review article)

This paper summarizes the history of and information on bovine herpesvirus type 4 (BoHV-4) from the first isolation to the most recent results. For almost twenty years BoHV-4 has been considered a typical herpes 'orphan' virus, which infects several species but causes no illness. The latest experiments revealed the close relationship of this virus with the immune system and other tissues. The virus was even considered as a possible candidate for a vector vaccine. BoHV-4 as a strange herpesvirus has several features which are not characteristic of other herpesviruses, such as several latency sites, persistence in serum, dividing cells necessary for virus replication, and the wide host range. In addition to describing the main features of the virion, replication, clinical signs, nomenclature problems, this review intends to concentrate on the new and strange results coming out from several laboratories worldwide. It is also suggested that because the virus combines several properties of various herpesvirus subfamilies and because of its close relationship with the immune system, it may deserve further attention as a representative of a potentially new genus within the Gammaherpesvirinae subfamily.     

Restriction of bovine herpesvirus 1 (BHV-1) growth in non-permissive cells beyond the expression of immediate early genes

Mouse BALB/3T3-A31-1-1 (A31) cells are non-permissive to bovine herpes virus-1 (BHV-1) but permissive to pseudorabies virus (PrV). The promoter activity of the immediate early gene of BHV-1 (BICP4) was very weak when compared with that of PrV in A31 cells. Infectious BHV-1 genomic DNA co-transfected into A31 cells with plasmids expressing BICP4 and BICP0 by a strong promoter failed to yield any progeny virus. Growth of BHV-1 in non-permissible A31 cells is restricted in many phases of the growth. The fact that expression of BICP4 and/or BICP0 in A31 cells does not improve the yield of progeny virus from infectious BHV-1 genomic DNA suggests that some more growth restrictions exist beyond the expression of BHV-1 immediate early proteins.     

Molecular Typing of a BHV-4 (Bovine herpesvirus 4) Field Isolate

A new BHV-4 (bovine herpesvirus 4) isolated from a case of bovine interdigital dermatitis was characterized by PCR and restriction enzyme analysis. To determine whether the new isolate (PR/1) belonged to BHV-4, DNA from infected cells was specifically amplified by PCR. We used a set of primers spanning a large 2.571 kb conserved region of the BHV-4 genome, including the 3 end of ORF1 (homologous to the EBV BVRF1 gene), ORF2 (homologous to the EBV BXRF1 gene), ORF3 (TK gene) and ORF4 (gH gene) 5 end, respectively. The identity of the amplified product was confirmed by HindIII restriction enzyme digestion and Southern hybridization. No product was observed from the DNA of other bovine herpesviruses tested. The restriction patterns of the PR/1 genome compared to DN 599, MOVAR 33/63 and LVR BHV-4 reference strains showed two kinds of differences, either related or not related to the prDNA (polyrepetitive DNA). Taken together, these data show that PR/1 is a new BHV-4. We would consider that the present report provides a scheme of work for diagnosis and typing of BHV-4 isolates.     

Bovine recombinant interleukin-2 augments immunity and resistance to bovine herpesvirus infection

The in vivo administration of bovine recombinant interleukin-2 (rIL-2) was evaluated in calves vaccinated and then challenged with bovine herpesvirus-1 (BHV-1). In Experiment 1, 24 calves were allotted to four groups: control; bovine rIL-2; BHV-1 vaccine (modified-live); and bovine rIL-2 + BHV-1 vaccine. Serum neutralizing antibody titers to BHV-1 were increased sixfold, and virus shedding was fourfold less in calves vaccinated and treated with rIL-2 (25 micrograms/kg, intramuscularly) when compared to calves that received vaccine only. Treatment with rIL-2 induced lymphokine-activated killer activity that was eliminated by pretreating effector cells with complement and a monoclonal antibody (B26A) specific for the sheep red blood cell receptor. The rIL-2 treatment in BHV-1-vaccinated calves increased the calves' ability to withstand a BHV-1 challenge. However, during treatment with rIL-2, calves developed diarrhea and mild fever that abated after IL-2 treatment was stopped. A second experiment was then conducted to determine a dose of rIL-2 that would enhance immunity to BHV-1 without causing adverse side effects. Twenty-five calves were allotted to five groups that received injections of rIL-2 at 0.0, 25.0, 2.5, 0.25, or 0.025 micrograms kg-1 day-1 for 5 days. All calves received a modified-live BHV-1 vaccine. Calves treated with 25.0 micrograms kg-1 day-1 showed similar adverse side effects as in the first experiment but all other calves were normal. Compared to control calves, those treated with 25.0, 2.5, and 0.25 micrograms kg-1 day-1 of rIL-2 had higher (P less than 0.05) serum antibody titers to BHV-1 and following challenge lower (P less than 0.05) BHV-1 titers in nasal secretions; additionally, clinical disease as evidenced by nasal and ocular discharge was less severe (P less than 0.05). In vitro cytotoxic responses against BHV-1-infected bovine kidney cells were increased (P less than 0.05) in calves treated with rIL-2 in a dose dependent manner. These data suggest that bovine rIL-2 at 2.5 to 0.25 micrograms/kg may be an effective adjuvant to immunization.     

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.     

Biological and molecular aspects of bovine herpesvirus 4 (BHV-4).

This review summarizes most recent information on the bovine cytomegalovirus BHV-4. The virus is not associated with clearly defined clinical entities in cattle. It can easily be isolated in tissue culture and has a broad host range. BHV-4 strains are rather similar in restriction enzyme analysis of their DNAs, the size of the pr DNAs, however, differs. The genome represents a gamma-herpesvirus. Because of its uniqueness BHV-4 is discussed as an appropriate vector.     

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.     

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.     

Characterisation of the fiber gene and partial sequence of the early region 4 of bovine adenovirus 2 (short communication)

The full sequence of the fiber gene and partial sequence of the putative 17 kD protein gene of bovine adenovirus-2 (BAdV-2) were determined. The size of the fiber gene of BAdV-2 proved to be 561 amino acids, of which the amino acids 37 to 385 form a typical shaft domain of 22 repetitive motifs. On the complementary strand, a gene homologous to the 17 kD protein coded in the E4 region of several human adenoviruses was found. The sequence analysis seems to confirm the presence of an intron in the sequenced part of the E4 region.     

Comparison of an enzyme-linked immunosorbent assay and a complement-fixation test for the detection of IgG to bovine herpesvirus type 4 (bovine cytomegalovirus)

An indirect ELISA for the detection of bovine immunoglobulin to bovine herpesvirus type 4 (BHV-4) was developed. Three methods of antigen preparation were compared. They included (1) BHV-4-infected Madin Darby bovine kidney (MDBK) cells treated with glycine and then frozen and thawed, (2) BHV-4-infected MDBK cells treated with glycine and then sonicated, and (3) BHV-4-infected MDBK cells treated with a detergent. The antigen preparation that gave the highest reactivity was the first method. We obtained serum samples from 178 cattle in the field and assayed the serum by ELISA and the complement-fixation (CF) test. Eighty-six percent (153) of the serum samples were positive by ELISA, and 70% (124) were positive by the CF test. The ELISA had a higher degree of sensitivity than did the CF test. Also, the ELISA was specific, and the prevalence of BHV-4-infection was more common in beef and dairy herds than previously recognized.     

A bovine herpesvirus (BHV-4) as passenger virus in ethmoidal tumours in Indian cattle

A herpesvirus was isolated from tumours of the ethmoidal mucosa in two of three head of cattle in the State of Kerala, India. The virus designated M40 was cytopathic for a variety of cultured bovine and porcine cells and it did not kill suckling mice or chicken embryos. Sera from tumour-bearing cattle and goats reacted with the M40 virus. Immunofluorescence tests with FITC-conjugated IgG from a bovine monospecific antiserum to bovine herpesvirus 4 (BHV-4) stained the M40 virus specific antigen in infected cells. Experimental infection of goats with the M40 virus did not result in development of tumours. This virus is therefore considered to represent a "passenger" virus. A great similarity was found between restriction patterns of DNAs extracted from M40 virus and the strain 66-P-347, a reference strain of the BHV-4 group.     

Bovine interleukin 2 (IL-2) production and activity on bovine and murine cell lines

Long-term growth of T cell cultures requires addition of Interleukin 2 (IL-2). In order to maintain bovine cultures, optimal conditions for bovine IL-2 production were defined using peripheral blood mononuclear cells (PBM). Irradiation and preculture enhanced IL-2 production possibly by reducing suppressor activity. IL-2 activity was also detected in Bovine Herpesvirus Type 1-stimulated cultures. Unlike mitogen-stimulated cultures, a wide variation in IL-2 activity was seen between supernatants produced by virus-stimulated cells from different animals indicating the clonal nature of antigen specific cells from individuals. Bovine IL-2-dependent cells used to quantitate IL-2 activity were characterized as: PNA, esterase negative, H4+ (anti Ia-like), B29+ (anti-pan T cell), and C5- (anti-monocyte). The observations that bovine IL-2 can maintain activated murine cells, CTLL-20 and HT-2, could lead to the replacement of rat IL-2 with bovine IL-2 in long-term murine cultures. Conditions described here result in large volumes of active medium.     

Bovine immune response to papillomatous digital dermatitis (PDD)-associated spirochetes is skewed in isolate reactivity and subclass elicitation

Papillomatous digital dermatitis (PDD) is a growing cause of lameness of dairy cattle worldwide. Farms with PDD-afflicted cows experience economic loss due to treatment costs, decreased milk production, lower reproductive efficiency and premature culling. Cows exhibit both humoral and cellular immune responses to PDD-associated spirochetes. This study was undertaken to further characterize the bovine humoral response to PDD-associated spirochetes. Forty-seven sera samples collected from cattle (Field cattle) on three different dairy operations in Iowa were analyzed. In addition, sera were obtained from six young steers (Test cattle) that received a mixed inoculum of four previously isolated Treponema phagedenis-like spirochetes (1A, 3A, 4A and 5B) on two separate occasions. Relative levels of total IgG, IgG1, IgG2 and IgM reactive to each individual spirochete were determined. Field cattle had a higher mean antibody response to 5B compared to the other isolates and T. phagedenis. Test cattle reacted most strongly with 4A following initial exposure, shifting to a greater reactivity with 5B and a reactivity profile similar to field cattle following secondary exposure. No measurable IgM was detected. IgG1 was produced predominately in all cattle. Low to moderate levels of total IgG reactivity to T. phagedenis occurred with sera from all cattle.